Combination therapy involving antibodies against claudin 18.2 for treatment of cancer

ABSTRACT

The present invention provides a combination therapy for effectively treating and/or preventing diseases associated with cells expressing CLDN18.2, including cancer diseases such as pancreatic cancer and metastases thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national stage entry of international applicationPCT/EP2014/000433, filed on Feb. 18, 2014, which claimed priority tointernational application PCT/EP2013/000505, filed on Feb. 20, 2013, theentire contents of which are incorporated herein by reference.

BACKGROUND

Pancreatic cancer is one of the most lethal cancers. The mortalityapproaches 100% because of the propensity for early metastatic spread,and because the disease is highly resistant to radiation andchemotherapy. Given that 27,000 new cases are diagnosed every year inNorth America and 68 000 in Europe there is an urgent need to developnovel treatment strategies to reduce the mortality of pancreatic cancerpatients.

The tight junction molecule Claudin 18 splice variant 2 (Claudin 18.2(CLDN18.2)) is a member of the claudin family of tight junctionproteins. CLDN18.2 is a 27.8 kDa transmembrane protein comprising fourmembrane spanning domains with two small extracellular loops. In normaltissues there is no detectable expression of CLDN18.2 by RT-PCR withexception of stomach. Immunohistochemistry with CLDN18.2 specificantibodies reveals stomach as the only positive tissue. CLDN18.2 is ahighly selective gastric lineage antigen expressed exclusively onshort-lived differentiated gastric epithelial cells. CLDN18.2 ismaintained in the course of malignant transformation and thus frequentlydisplayed on the surface of human gastric cancer cells. Moreover, thispan-tumoral antigen is ectopically activated at significant levels inesophageal, pancreatic and lung adenocarcinomas.

The chimeric IgG1 antibody IMAB362 which is directed against CLDN18.2has been developed by Ganymed Pharmaceuticals AG. IMAB362 recognizes thefirst extracellular domain (ECD1) of CLDN18.2 with high affinity andspecificity. IMAB362 does not bind to any other claudin family memberincluding the closely related splice variant 1 of Claudin 18 (CLDN18.1).IMAB362 shows precise tumor cell specificity and bundles fourindependent highly potent mechanisms of action. Upon target bindingIMAB362 mediates cell killing by ADCC, CDC and induction of apoptosisinduced by cross linking of the target at the tumor cell surface anddirect inhibition of proliferation. Thus, IMAB362 lyses efficientlyCLDN18.2-positive cells, including human gastric cancer cell lines invitro and in vivo.

The toxicity and PK/TK profile of IMAB362 has been thoroughly examinedin mice and cynomolgus monkeys including dose range finding studies,28-day repeated dose toxicity studies in cynomolgus and a 3-monthrepeated dose toxicity study in mice. In both mice (longest treatmentduration weekly administration for 3 months, highest dose levels 400mg/kg) and cynomolgus monkeys (up to 5 weekly applications of up to 100mg/kg) repeated doses of IMAB362 i.v. are well tolerated. No signs ofsystemic or local toxicity are induced. Specifically, no gastrictoxicity has been observed in any toxicity study. IMAB362 does notinduce immune activation and cytokine release. No adverse effects onmale or female reproductive organs were recorded. IMAB362 does not bindto tissues lacking the target. Biodistribution studies in mice indicatethat the reason for lack of gastric toxicity is most likelycompartimentalization of tight junctions at the luminal site in healthygastric epithelia, which appears to impair accessibility of the IMAB362epitope profoundly.

IMAB362 is in early clinical testing. A phase I clinical study has beenconducted in human. 5 dose cohorts (33 mg/m², 100 mg/m², 300 mg/m², 600mg/m², 1000 mg/m²) of 3 patients each have received a single intravenousadministration of IMAB362 and have been observed for 28 days. IMAB362was very well tolerated, with no relevant safety observation in thepatients. In one patient all measured tumor markers decreasedsignificantly within 4 weeks after treatment. In an ongoing phase IIaclinical study IMAB362 is given repetitively.

Here we present data demonstrating that chemotherapeutic agents canstabilize or increase expression of CLDN18.2 on the surface ofpancreatic cancer cells resulting in an enhanced drugability of CLDN18.2by an anti-CLDN18.2 antibody such as IMAB362. A synergistic effect of ananti-CLDN18.2 antibody such as IMAB362 with particular chemotherapeuticregimens, in particular chemotherapeutic regimens used for pancreaticcancer treatment was observed. Human cancer cells pre-treated withchemotherapy are more susceptible to antibody-induced target-specifickilling. In mouse tumor models, tumor control with an anti-CLDN18.2antibody plus chemotherapy is superior to that with an anti-CLDN18.2antibody as single agent.

SUMMARY OF THE INVENTION

The present invention generally provides a combination therapy foreffectively treating and/or preventing diseases associated with cellsexpressing CLDN18.2, including cancer diseases such as gastric cancer,esophageal cancer, pancreatic cancer, lung cancer such as non small celllung cancer (NSCLC), ovarian cancer, colon cancer, hepatic cancer,head-neck cancer, and cancer of the gallbladder and metastases thereof,in particular gastric cancer metastasis such as Krukenberg tumors,peritoneal metastasis and lymph node metastasis. Particularly preferredcancer diseases are pancreatic cancer and the metastases thereof.

In one aspect, the present invention provides a method of treating orpreventing pancreatic cancer in a patient comprising administering tothe patient (i) an antibody having the ability of binding to CLDN18.2and (ii) an agent stabilizing or increasing expression, i.e. the level,of CLDN18.2. Expression of CLDN18.2 is preferably at the cell surface ofa cancer cell. The agent stabilizing or increasing expression ofCLDN18.2 may be administered prior to, simultaneously with or followingadministration of the antibody having the ability of binding toCLDN18.2, or a combination thereof.

The agent stabilizing or increasing expression of CLDN18.2 may be acytotoxic and/or cytostatic agent. In one embodiment, the agentstabilizing or increasing expression of CLDN18.2 comprises an agentwhich induces a cell cycle arrest or an accumulation of cells in one ormore phases of the cell cycle, preferably in one or more phases of thecell cycle other than the G1-phase such as the S-phase, G2-phase, or acombination thereof or a combination of the S-phase or the G2-phase withthe G1-phase. The agent stabilizing or increasing expression of CLDN18.2may comprise an agent selected from the group consisting of nucleosideanalogs, platinum compounds, camptothecin analogs and taxanes, prodrugsthereof, salts thereof, and combinations thereof. The nucleoside analogmay be selected from the group consisting of gemcitabine,5-fluorouracil, prodrugs thereof and salts thereof. The platinumcompound may selected from the group consisting of oxaliplatin,cisplatin, prodrugs thereof and salts thereof. The camptothecin analogmay be selected from the group consisting of irinotecan, topotecan,prodrugs thereof and salts thereof. The taxane may be selected from thegroup consisting of paclitaxel, docetaxel, prodrugs thereof and saltsthereof. The agent stabilizing or increasing expression of CLDN18.2 maycomprise an agent selected from the group consisting of gemcitabine,5-fluorouracil, oxaliplatin, irinotecan, paclitaxel, prodrugs thereof,salts thereof and combinations thereof. The agent stabilizing orincreasing expression of CLDN18.2 may comprise a combination ofoxaliplatin and 5-fluorouracil or prodrugs thereof, a combination ofcisplatin and 5-fluorouracil or prodrugs thereof, a combination of atleast one taxane and oxaliplatin, a combination of at least one taxaneand cisplatin, a combination of at least one taxane and 5-fluorouracilor prodrugs thereof, or a combination of at least one camptothecinanalog and 5-fluorouracil or prodrugs thereof. The agent stabilizing orincreasing expression of CLDN18.2 may comprise a combination ofgemcitabine and oxaliplatin, a combination of gemcitabine and cisplatin,a combination of gemcitabine and carboplatin or a combination ofoxaliplatin, 5-fluorouracil or prodrugs thereof and irinotecan.Accordingly, the method of the invention may comprise administering acombination of gemcitabine and oxaliplatin, a combination of gemcitabineand cisplatin, a combination of gemcitabine and carboplatin or acombination of oxaliplatin, 5-fluorouracil or prodrugs thereof andirinotecan. In one embodiment, the method of the invention comprisesadministering folinic acid, 5-fluorouracil or prodrugs thereof,irinotecan and oxaliplatin. The agent stabilizing or increasingexpression of CLDN18.2 may comprise an agent inducing immunogenic celldeath. The agent inducing immunogenic cell death may compriseoxaliplatin.

In a further aspect, the present invention provides a method of treatingor preventing cancer in a patient comprising administering to thepatient (i) an antibody having the ability of binding to CLDN18.2 and(ii) gemcitabine. In one embodiment, the cancer is selected from thegroup consisting of gastric cancer, esophageal cancer, pancreaticcancer, lung cancer, ovarian cancer, colon cancer, hepatic cancer,head-neck cancer, cancer of the gallbladder and the metastasis thereof.The cancer disease may be a Krukenberg tumor, peritoneal metastasisand/or lymph node metastasis. In one embodiment, the cancer is anadenocarcinoma, in particular an advanced adenocarcinoma. In oneembodiment, the cancer is pancreatic cancer.

In one embodiment, the method of the invention further comprisesadministering an agent stimulating γδ T cells. In one embodiment, the γδT cells are Vγ9Vδ2 T cells. In one embodiment, the agent stimulating γδT cells is a bisphosphonate such as a nitrogen-containing bisphosphonate(aminobisphosphonate). In one embodiment, the agent stimulating γδ Tcells is selected from the group consisting of zoledronic acid,clodronic acid, ibandronic acid, pamidronic acid, risedronic acid,minodronic acid, olpadronic acid, alendronic acid, incadronic acid andsalts thereof. In one embodiment, the agent stimulating γδ T cells isadministered in combination with interleukin-2.

The method of the invention may further comprise administering at leastone further chemotherapeutic agent which may be a cytotoxic agent.

The antibody having the ability of binding to CLDN18.2 may bind tonative epitopes of CLDN18.2 present on the surface of living cells. Inone embodiment, the antibody having the ability of binding to CLDN18.2binds to the first extracellular loop of CLDN18.2. In one embodiment,the antibody having the ability of binding to CLDN18.2 mediates cellkilling by one or more of complement dependent cytotoxicity (CDC)mediated lysis, antibody dependent cellular cytotoxicity (ADCC) mediatedlysis, induction of apoptosis and inhibition of proliferation. In oneembodiment, the antibody having the ability of binding to CLDN18.2 is amonoclonal, chimeric or humanized antibody, or a fragment of anantibody. In one embodiment, the antibody mediates cell killing whenbound to cellular CLDN18.2, in particular to CLDN18.2 expressed by cellson their cell surface, wherein the cells are preferably cancer cells,such as cells of the cancers described herein. In one embodiment, theantibody having the ability of binding to CLDN18.2 is an antibodyselected from the group consisting of (i) an antibody produced by and/orobtainable from a clone deposited under the accession no. DSM ACC2737,DSM ACC2738, DSM ACC2739, DSM ACC2740, DSM ACC2741, DSM ACC2742, DSMACC2743, DSM ACC2745, DSM ACC2746, DSM ACC2747, DSM ACC2748, DSMACC2808, DSM ACC2809, or DSM ACC2810, (ii) an antibody which is achimerized or humanized form of the antibody under (i), (iii) anantibody having the specificity of the antibody under (i) and (iv) anantibody comprising the antigen binding portion or antigen binding site,in particular the variable region, of the antibody under (i) andpreferably having the specificity of the antibody under (i). In oneembodiment, the antibody is coupled to a therapeutic agent such as atoxin, a radioisotope, a drug or a cytotoxic agent.

In one embodiment, the method of the invention comprises administeringthe antibody having the ability of binding to CLDN18.2 at a dose of upto 1000 mg/m². In one embodiment, the method of the invention comprisesadministering the antibody having the ability of binding to CLDN18.2repeatedly at a dose of 300 to 600 mg/m².

According to the invention, CLDN18.2 preferably has the amino acidsequence according to SEQ ID NO: 1.

In one embodiment, the cancer described herein is CLDN18.2 positive. Inone embodiment, cancer cells of the cancer described herein are CLDN18.2positive. In one embodiment, cancer cells of the cancer described hereinexpress CLDN18.2 on their cell surface.

In one embodiment, the pancreatic cancer described herein comprisesprimary cancer, advanced cancer or metastatic cancer, or a combinationthereof such as a combination of pancreatic primary cancer andmetastatic cancer. In one embodiment, the methods of the invention arefor the simultaneous treatment of primary cancer and metastatic cancersuch as pancreatic primary cancer and pancreatic metastatic cancer. Inone embodiment, the metastatic cancer comprises metastasis to the lymphnodes, ovary, liver or lung, or a combination thereof. In oneembodiment, the pancreatic cancer comprises cancer of the pancreaticduct. In one embodiment, the pancreatic cancer comprises anadenocarcinoma or carcinoma, or a combination thereof. In oneembodiment, the pancreatic cancer comprises a ductal adenocarcinoma, amucinous adenocarcinoma, a neuroendocrine carcinoma or an acinic cellcarcinoma, or a combination thereof. In one embodiment, the pancreaticcancer is partially or completely refractory to gemcitabine treatmentsuch as gemcitabine monotherapy. In one embodiment, preventingpancreatic cancer comprises preventing a recurrence of pancreaticcancer.

In one embodiment, the patient to be treated according to the inventionhad a surgery for pancreatic cancer. In one embodiment, the patient hasa precancerous pancreatic lesion, in particular a precancerouspancreatic lesion comprising a beginning malignant histological changein the pancreatic ducts. In these embodiments, the methods of theinvention preferably aim at preventing the development of malignantpancreatic cancer.

In a further aspect, the present invention provides a medicalpreparation for treating or preventing pancreatic cancer comprising (i)an antibody having the ability of binding to CLDN18.2 and (ii) an agentstabilizing or increasing expression of CLDN18.2. The medicalpreparation of the present invention may further comprise an agentstimulating γδ T cells. The antibody having the ability of binding toCLDN18.2 and the agent stabilizing or increasing expression of CLDN18.2,and optionally the agent stimulating γδ T cells, may be present in themedical preparation in a mixture or separate from each other. Themedical preparation may be present in the form of a kit comprising afirst container including the antibody having the ability of binding toCLDN18.2 and a second container including the agent stabilizing orincreasing expression of CLDN18.2, and optionally a container includingthe agent stimulating γδ T cells. The medical preparation may furtherinclude printed instructions for use of the preparation for treatment orprevention of pancreatic cancer, in particular for use of thepreparation in a method of the invention. Different embodiments of themedical preparation, and, in particular, of the antibody having theability of binding to CLDN18.2, the agent stabilizing or increasingexpression of CLDN18.2 and the agent stimulating γδ T cells are asdescribed above for the methods of the invention.

In a particular aspect, the present invention provides a medicalpreparation comprising (i) an antibody having the ability of binding toCLDN18.2, and (ii) gemcitabine. The medical preparation of the presentinvention may further comprise an agent stimulating γδ T cells. Theantibody having the ability of binding to CLDN18.2 and gemcitabine, andoptionally the agent stimulating γδ T cells, may be present in themedical preparation in a mixture or separate from each other. Themedical preparation may be for treating or preventing cancer such aspancreatic cancer. The medical preparation may be present in the form ofa kit comprising a first container including the antibody having theability of binding to CLDN18.2 and a second container includinggemcitabine, and optionally a container including the agent stimulatingγδ T cells. The medical preparation may further include printedinstructions for use of the preparation for treatment or prevention ofcancer such as pancreatic cancer, in particular for use of thepreparation in a method of the invention. Different embodiments of themedical preparation, and, in particular, of the antibody having theability of binding to CLDN18.2, the agent stabilizing or increasingexpression of CLDN18.2 and the agent stimulating γδ T cells are asdescribed above for the methods of the invention.

The present invention also provides the agents described herein such asthe antibody having the ability of binding to CLDN18.2 and/or the agentstabilizing or increasing expression of CLDN18.2 for use in the methodsdescribed herein. For example, the present invention also provides theantibody having the ability of binding to CLDN18.2 for administration inconjunction with an agent stabilizing or increasing expression ofCLDN18.2 such as gemcitabine, and optionally an agent stimulating γδ Tcells.

Other features and advantages of the instant invention will be apparentfrom the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a lentiviral vector used for transduction of pancreascancer cell lines. Human CLDN18.2 was cloned downstream of the EF1αpromoter. The expression cassette is integrated between the longterminal repeats (5′ and 3′-LTR) which enable packaging and reversetranscription of the viral mRNA. RSV: rous sarcoma virus allows Tatindependent production of viral mRNA. Amp: Ampicillin resistance gene.PGKp: Promotor of blasticidin. WPRE: woodchuck posttranscriptionalregulatory element; enhances transgene expression. LTR: long terminalrepeat, allows viral packaging. SV40A allows transcriptional terminationand polyadenylation of mRNA. pUC: bacterial vector backbone. Bla:promoter of ampicillin.

FIG. 2 shows a metastasis analysis of pancreas cells in mouse lung.Dissection scheme of mouse lungs after i.v. injection of mice withpancreas cancer cells.

FIGS. 3A and 3B show a CLDN18.2 expression in normal and cancerouspancreatic tissues. Staining of normal pancreas formalin fixed paraffinembedded (FFPE) tissue (FIG. 3A) and pancreas adenocarcinoma tissue(FIG. 3B) with the monoclonal murine 35-22A antibody (0.2 μg/ml).Haematoxylin counterstaining (2:00 min). Magnification 200×.

FIGS. 4A, 4B, and 4C show a CLDN18.2 expression in normal andprecancerous pancreatic tissues. 43-14A staining of various precancerousstructures (FIG. 4A) normal and PanIN1; (FIG. 4B) PanIN2; (FIG. 4C)PanIN3. Magnification 200×.

FIG. 5 shows the correlation between CLDN18.2 signal intensity andamount of positive tumor cells for the analyzed pancreas primary tumors(pilot study). Each dot represents a pancreatic primary cancer caseanalyzed by staining FFPE sections using the monoclonal, murine 35-22Aantibody (0.2 μg/ml). The dashed line marks the 10% value.

FIGS. 6A and 6B show expression of CLDN18.2 in primary and metastaticpancreatic tumor tissue (pilot study). Staining of FFPE tissue sections(3 μm) using the murine, monoclonal 35-22A antibody of (FIG. 6A)adenocarcinoma primary tumor and (FIG. 6B) lymph node metastasis.Haematoxylin (Mayers) counterstained.

FIG. 7 shows the correlation between CLDN18.2 signal intensity andamount of positive tumor cells for the analyzed pancreas primary tumors(main study). Each dot represents a pancreatic ductal adenocarcinomaprimary tumor (filled circle) or a neuroendocrine primary tumor (opencircle) case analyzed by staining FFPE sections using monoclonal murine43-14A antibody (0.2 μg/ml).

FIG. 8 shows the correlation between CLDN18.2 signal intensity andamount of positive tumor cells for the analyzed pancreas metastases.Each dot represents a pancreatic lymph node (filled circle) or liver(open circle) metastasis case analyzed by staining FFPE sections usingthe monoclonal, murine 43-14A antibody (0.2 μg/ml). The dashed linemarks the 10% value.

FIGS. 9A, 9B, 9C, 9D, 9E and 9F show expression of CLDN18.2 in primaryand metastatic pancreatic tumor tissue. Staining of FFPE tissue sections(3 μm) using the murine, monoclonal 43-14A antibody on (FIGS. 9A, 9C and9E) adenocarcinoma primary tumor and (FIGS. 9B, 9D and 9F) lymph nodemetastasis. The sections were counterstained using Mayers Haematoxylin.

FIG. 10 shows expression of CLDN18.2 in matched pancreatic primary tumorand lymph node metastatic tissues (graphical analysis).

FIGS. 11A, 11B and 11C show expression of CLDN18.2 in matched pancreaticprimary tumor and metastatic tissues. Staining of FFPE tissue sections(3 μm) of (FIG. 11A) primary adenocarcinoma, (FIG. 11B) liver metastasisand (FIG. 11C) lymph node metastasis, using the murine, monoclonal43-14A antibody. The sections were counterstained using MayersHaematoxylin. 200× magnification.

FIGS. 12A, 12B, 12C and 12D show CLDN18.2 mRNA levels in pancreaticcarcinoma cell lines. FIG. 12A shows Q-PCR expression analyses ofdifferent pancreas CA cell lines, the lentivirally transduced (LVT) celllines (gray bars), the stomach cancer cell line KATO-III (positivecontrol) and the breast cancer cell line SKBR-3 (negative control).CLDN18.2 transcripts were amplified using gene specific primers.Endogenous cell lines showing a relative expression level above 1×10⁵were scored as CLDN18.2 positive (hatched bars). NTC: H₂O controlsample. Error bars: Mean+SD. (FIGS. 12B-12D) Passage-dependent CLDN18.2expression analyses in Patu8988S (FIG. 12B), Panc05.04 (FIG. 12C) andthe indicated LVT cell lines (FIG. 12D). Passage number is indicatedbelow each bar.

FIGS. 13A and 13B show CLDN18.2 protein levels in cell lysates ofpancreatic carcinoma cell lines. Proteins were separated on a 12.5%SDS-PAGE. Western Blot analysis was performed using a CLDN18 antibodydetecting the C-terminal of CLDN18.1 and CLDN18.2 (Zymed-MID) and usinga loading control antibody detecting β-actin. Exposure times of 140 sec(Pierce SuperSignal West Dura) and 20 sec (Pierce SuperSignal West Pico)were used respectively. FIG. 13A shows detection of CLDN18 in pancreascell line lysates, the positive control (HEK293-p740) and the negativecontrol cell lysates (SKBR-3). FIG. 13B shows CLDN18.2 expressioncompared between non-transduced parental cell lysates and lentivirallytransduced (LVT) cell line lysates. Patu8988S and SKBR-3 were added aspositive and negative control, respectively.

FIGS. 14A, 14B, 14C, 14D, 14E, 14F, 14G, 14H, 14I, 14J, 14K, 14L, 14M,14N, 14O, 14P, 14Q, 14R and 14S show detection and cellular localizationof CLDN18 expression in pancreatic cancer cell lines. Staining ofpancreatic cancer cell lines grown on cover slips. Antibody: 35-22A (20×magnification, the exposure time is indicated below each picture) DAPIwas used to stain the nuclei (blue). (FIG. 14A: AsPC1; FIG. 14B: BxPC3;FIG. 14C: CFPAC; FIG. 14D: DANG; FIG. 14E: HPAF-II; FIG. 14F: HUP-T3;FIG. 14G: HUP-T4; FIG. 14H: KCI-MOH; FIG. 14I: Panel; FIG. 14J:Panc05.04; FIG. 14K: Panc02.04; FIG. 14L: Panc04.03; FIG. 14M: Patu8902;FIG. 14N: Patu8988S; FIG. 14O: Su86.86: FIG. 14P: Suit-2; FIG. 14Q:SW-1990; FIG. 14R: YAPC; FIG. 14S: gastric cancer control cell lineKATO-III).

FIGS. 15A, 15B, 15C, 15D, 15E, 15F, 15G and 15H show detection andcellular localization of CLDN18 expression in CLDN18.2 transducedpancreatic cancer cell lines. CLDN18 detection in the lentivirallytransduced (LVT) pancreatic cancer cell lines using 35-22A antibodyafter fixation and permeabilization. Alexa488 or Alexa555 labeledsecondary antibodies were used for detection. FIG. 15A: BxPC3-LVT; FIG.15B: CAPAN1-LVT; FIG. 15C: DANG-LVT; FIG. 15D: HPAC-LVT; FIG. 15E:MiaPaCa2-LVT; FIG. 15F: Patu8902-LVT; FIG. 15G: Suit-2-LVT; FIG. 15H:YAPC-LVT.

FIGS. 16A, 16B, 16C, 16D, 16E, 16F, 16G, 16H, 16I, 16J, 16K and 16L showbinding of IMAB362 to the cell surface of CLDN18.2 positive pancreas CAcell lines (pharmacodynamics). IF analysis of pancreatic cancer celllines (FIGS. 16A, 16B, 16D and 16E), lentivirally transduced pancreascell lines (FIGS. 16G-16L) and KATO-III gastric cancer control cells(FIGS. 16C and 16F) expressing CLDN18.2. Cells were stained with IMAB362under native conditions (FIGS. 16D-16E) and for comparison with 35-22Aafter fixation and permeabilization of the cells (FIGS. 16A-16C). DAPIwas used to stain the nuclei. Exposure times are indicated in eachpanel. FIG. 16G: BxPC3-LVT; FIG. 16H: CAPAN1-LVT; FIG. 16I: DANG-LVT;FIG. 16J: MiaPaCa2-LVT; FIG. 16K: Patu8902-LVT; FIG. 16L: Suit2-LVT.

FIGS. 17A, 17B, 17C, 17D, 17E, 17F, 17G, 17H, 17I, 17J, 17K, 17L and 17Mshow CLDN18.2 expression in xenograft tumors of different cell lines.Expression of CLDN18.2 in CAPAN1-LVT (FIGS. 17A and 17B), BxPC3-LVT(FIGS. 17C and 17D), PATU8988S-LVT (FIGS. 17E and 17F), MiaPaCa2-LVT(FIGS. 17G and 17H), YAPC-LVT (FIGS. 17J and 17K) and DANG-LVT (FIGS.17L and 17M) xenograft tumors. Tissue staining was performed withZymed-MID antibody. Magnification lens 10× (FIGS. 17A, 17C, 17E, 17G,17J and 17L) and 20× (FIGS. 17B, 17D, 17F, 17H, 17K and 17M).

FIGS. 18A, 18B, 18C, 18D, 18E and 18F show an engraftment check ofSuit-2 and MiaPaCa2 pancreatic cancer cell lines. Cells were injectedinto the tail vein of nude mice. Animals were sacrificed 45 (FIG. 18A),52 (FIG. 18B), 59 (FIG. 18C) days after Suit-2 (FIGS. 18A-18C)application or 59 (FIG. 18D), 66 (FIG. 18E), 73 (FIG. 18F) days afterMiaPaCa2 (FIGS. 18D-18F) injection. Lungs were prepared and stained withMHC class I antibodies (anti-human MHC I, clone EPR1394Y) to detect thehuman cells in mouse tissues.

FIGS. 19A and 19B show a metastasis engraftment analysis of Patu8988S.Patu8988S cells were i.v. injected with 1×10⁶ or 2×10⁶ cells in Nu/Numice and lungs (FIG. 19A) and livers (FIG. 19B) of the mice wereisolated at different time points as indicated below the x-axis. Tocalculate the % of human DNA present in each tissue preparation, astandard curve was prepared by mixing human and mouse DNA and preparing7×5-fold dilutions resulting in 100% (1)-0.0064% (7) human DNA.

FIGS. 20A, 20B, 20C, 20D, 20E, 20F, 20G and 20H show an IHC analysis ofPatu8988S metastasis in mouse lung tissues. Mice injected in their tailveins with Patu8988S cells were sacrificed at different time points(FIGS. 20A-20D=70 days, FIGS. 20E-20H=86 days) and lung tissues wereisolated and stained with an MHC-I (EPR1394Y) antibody (FIGS. 20A, 20B,20E and 20F) diluted 1:1000 or with anti-Claudin18 (Zymed-Mid) (FIGS.20C, 20D, 20G and 20H) at 0.2 μg/ml. Magnifications: FIGS. 20A, 20C, 20Eand 20G=10× and FIGS. 20B, 20D, 20F and 20H=20×.

FIG. 21 shows an IMAB362 mediated apoptosis of gemcitabine treatedpancreas tumor cells. Apoptosis induced by cross-linking of CLDN18.2 onBxPC3˜CLDN18 after 48 hours. BxPC3˜CLDN18 were cultivated in medium ormedium+100 ng/ml gemcitabine. Apoptotic cell fraction of mononuclearcells were shifted. Similar shifts were obtained by incubation of tumorcells with Camptotecin.

FIGS. 22A, 22B, 22C, 22D, 22E, 22F, 22G and 22H show potency ofIMAB362-induced ADCC activity on pancreatic cancer cells. FIG. 22A showsADCC performed with CLDN18.2 positive pancreatic cancer cell lines usingPBMCs of different donors. FIGS. 22B-22F show ADCC performed with LVTpancreas cell lines ectopically expressing CLDN18.2 and thecorresponding parental cells. FIG. 22G shows a dot plot.

FIGS. 23A, 23B, 23C, 23D and 23E show potency of IMAB362-induced CDCactivity on pancreatic cancer cells. FIG. 23A shows a CDC performed withhealthy human serum pool as complement source, IMAB362 and CLDN18.2positive pancreas CDOK1-p740 control cells in 4 independent experiments.FIG. 23B shows a CDC performed with CLDN18.2 positive (Patu8988S, DANG,Panc05.04) and CLDN18.2 negative (CAPAN1, Suitt, BxPC3, YAPC) pancreascell lines. FIG. 23C shows a CDC with ectopically expressing LVT celllines. FIG. 23D shows a dot plot showing IMAB362 concentration causinghalf maximum lysis rates (EC50) on pancreatic cancer cell lines. FIG.23E shows maximum killing rates obtained with IMAB362 on pancreaticcancer cell lines.

FIGS. 24A and 24B show the effect of IMAB362 treatment on subcutaneousMiaPaCa2-LVT xenografts. MiaPaCa2-LVT xenograft tumors were inoculatedby injection of 1e7 MiaPaCa2-LVT cells subcutaneous into the flank of 15female Hsd:Athymic Nude-Foxn1nu mice for each treatment group. On thethird day after tumor cell injection, treatment was initiated with 200μg IMAB362 or controls respectively. Treatment was continued semi-weeklywith alternating i.p. and i.v. injection until animals were sacrificed.FIG. 24A shows the effect of IMAB362 treatment on tumor growth. The sizeof s.c. tumors was measured twice weekly (mean+SEM). FIG. 24B showsKaplan-Meier survival plots. Mice were sacrificed when tumor reached avolume of 1400 mm³ or tumor became ulcerous.

FIGS. 25A and 25B show IMAB362 treatment of subcutaneous BxPC3-LVTxenografts. BxPC3-LVT xenograft tumors were inoculated by injection of1e7 BxPC3-LVT cells subcutaneous into the flank of 15 female Hsd:AthymicNude-Foxn1nu mice for each treatment group. On the third day after tumorcell injection, treatment was initiated with 200 μg IMAB362 or controlsrespectively. Treatment was continued semi-weekly with alternating i.p.and i.v. injection until animals were sacrificed. FIG. 25A shows theeffect of IMAB362 treatment on tumor growth. The size of s.c. tumors wasmeasured twice weekly (mean+SEM, * p<0.05). FIG. 25B shows Kaplan-Meiersurvival plots. Mice were sacrificed, when tumor reached a volume of1400 mm³ or tumor became ulcerous.

FIGS. 26A and 26B show the effect of IMAB362 treatment on growth ofSuit2-LVT pancreas metastasis. 2×10⁶ Suit2-LVT tumor cells were injectedintravenously into the tail vein of 12 female Hsd:Athymic Nude-Foxn1numice per treatment group. On the third day after tumor cell injection,treatments were initiated with 200 μg IMAB362, 200 μg isotype control orwith an equal volume of PBS. Animals were sacrificed on day 42 postgraft. FIG. 26A shows a qPCR analysis (mean of 2-4 reactions per sample)determining the percentage of human DNA present in the mouse lungsamples. FIG. 26B shows the percentage of human cells covering the mouselung surface was determined by planimetry. Human cells wereimmunohistochemically stained in tissue sections with anti-humanMHC-class I antibodies. * p<0.05 (Kruskal-Wallis test). Error bars:mean±SD.

FIGS. 27A, 27B, 27C, 27D and 27E show Q-PCR and IHC analyses ofPatu8988S lung metastasis. 2×10⁶ Patu8988S cells were injected permouse. Animals were sacrificed after 65 days. Open circles: micesacrificed after 63 days. FIG. 27A shows mice that were treated with 200μg IMAB362 semi-weekly or saline control. Amount of human DNA (ng)detected in with Q-PCR, which was calculated from the Ct values. FIG.27B shows a Q-PCR experiment repeat as described in Figure A. Here thepercentage of human DNA present in mouse DNA was calculated from the Ctvalues. FIG. 27C shows mice that were treated with IMAB362 and isotypecontrol antibody (rituximab). The percentage of human DNA present in themouse lungs was calculated from the Ct values. For the IMAB362 group,one outlier was detected (open triangle). The significance is indicatedby including or excluding the outlier values. (FIGS. 27D and 27E) sameexperiment as in FIG. 27C. Here surface of the metastasis was determinedusing the Image J program. Dot plots show the significance of IMAB362inhibition including (FIG. 27D) or excluding (FIG. 27E) the outliervalue. P-value: unpaired t-test. Error bars±SD

FIG. 28 shows dose response curves for gemcitabine. Pancreas cancer celllines show very different sensitivity for gemcitabine. Cell lines wereexposed for 4 days with different concentrations of gemcitabine andinhibition of proliferation analysed via viability assay.

FIG. 29 shows dose response curves for oxaliplatin. Pancreas cancer celllines show very different sensitivity for oxaliplatin. Cell lines wereexposed for 4 days with different concentrations of oxaliplatin andinhibition of proliferation analysed via viability assay.

FIGS. 30A and 30B show the effect of treatment with chemotherapeuticagents on CLDN18.2 expression (RNA). RNA of untreated, Gem (1 ng/ml) orGemOx (Gem 1 ng/ml+Ox 10 ng/ml) pretreated DANG (2 days) (FIG. 30A) orPatu8988S (FIG. 30B) cells 3 days pretreated with Gem (10 ng/ml or GemOx(Gem 10 ng/ml+Ox 100 ng/ml). RNA was converted to cDNA. CLDN18.2transcript level was analyzed in quantitative real-time PCR. Results areshown as relative units in comparison to transcript level of housekeeping gene HPRT.

FIG. 31 shows effect of chemotherapy on CLDN18.2 protein level inpancreatic carcinoma cells. Protein from total cell lysates of untreated(med), Gem (1 ng/ml) or GemOx (Gem 1 ng/ml+Ox 10 ng/ml) pretreated DANG(A) or Patu8988S (B) cells were analyzed for CLDN18.2 expressiondetected with Zymed C-term polyclonal antisera. Actin was used to showequal loading of proteins.

FIG. 32 shows FACS analysis of CLDN18.2 cell surface expression. CLDN18expression (filled histogram) of medium cultivated (left) and Gemtreated (right) Patu8988S is shown in an overlay compared to Isotyp Co.Patu8988S are treated with gemcitabine (10 ng/ml)) for 3 days.

FIGS. 33A and 33B shows cell cycle analyses of DANG cells treated or notwith either gemcitabine (Gem; 2 ng/ml) or gemcitabine+oxaliplatin(GemOx; 1 ng/ml+10 ng/ml) for two days. (33A) Gemcitabine treatmentleads to cell cycle arrest of cells in S-Phase. The area of each bar isdivided to indicate the percentage of cells in G0/G1, S and G2 phase.(33B) Western blot analyses showed upregulation of CLDN18 aftertreatment with Gem.

FIGS. 34A, 34B and 34C show influence of gemcitabine on cell cycle (FIG.34A) and CLDN18.2 expression (FIGS. 34B and 34C) in Patu8988S cells.Patu8988S cells were either untreated or treated with gemcitabine (10ng/ml) for 2 days. In FIG. 34A, the area of each bar is divided toindicate the percentage of cells in G0/G1, S and G2 phase. The densityof CLDN18.2 (x-axis) was plotted against the cell number (y-axis). InFIG. 34B, CLDN18.2 expression of untreated (dotted line) versus Gemtreated (solid line) is blotted. In FIG. 34C, CLDN18.2 expression of gemtreated Patu8988S cells in G0/G1 phase (dotted line) versus cells in Sphase (solid line) is shown.

FIGS. 35A, 35B, 35C, 35D and 35E show effect of chemotherapy on gastriccancer cells. Cultivation of Kato III cells for 96 hours leads to a cellcycle arrest in the G0/G1-phase (FIGS. 35A and 35C) and downregulationof CLDN18.2 (FIG. 35D). Cytostatic compounds resulting in a cell cyclearrest in different phases of the cell cycle stabilizeCLDN18.2-expression (FIG. 35D).

FIG. 36 shows effect of chemotherapy on gastric cancer cells. Cytostaticcompounds resulting in a cell cycle arrest in different phases of thecell cycle (S/G2-phase (Irinotecan) or G2-phase (Docetaxel)). The areaof each bar is divided to indicate the percentage of cells in G0/G1, Sand G2 phase.

FIGS. 37A and 37B show dose response curves for IMAB362 mediated ADCCafter chemotherapy treatment of DANG. FIG. 37A shows dose responsecurves of one representary donor after pretreatment of DANG pancreascancer cells with Gem or GemOx for 40 h. FIG. 37B shows EC50 values(mean) for IMAB362 mediated ADCC. P-values: unpaired t-test.

FIGS. 38A, 38B, 38C and 38D show effect of chemotherapy on gastriccancer cells. In FIG. 38A, cells treated with Irinotecan, Docetaxel orCisplatin exhibit a lower level of viable cells compared to mediumcultivated target cells. In FIG. 38B, CLDN18.2 expression in cellstreated with Irinotecan, Docetaxel or Cisplatin is increased compared tomedium cultivated cells. In FIGS. 38C and 38D, treatment of cells withIrinotecan, Docetaxel or Cisplatin augments the potency of IMAB362 toinduce ADCC.

FIG. 39 shows the influence of chemotherapeutic agents on IMAB362mediated CDC of MiaPaCa2-LVT cells. Dose response curves of 2independent assays. MiaPaCa2-LVT were cultivated in medium, Gem (10ng/ml) or GemOx (10 ng/ml Gem+100 ng/ml Ox) for 70 h.

FIG. 40 shows the effects of chemotherapy on IMAB362-induced CDC

FIGS. 41A and 41B show the effect of IMAB362 treatment combined with Gemor GemOx on BxPC3-LVT xenografts. BxPC3-LVT xenograft tumors wereinoculated by injection of 8.5e6 BxPC3-LVT cells subcutaneous into theflank of 10 female Hsd:Athymic Nude-Foxn1nu mice for each treatmentgroup. On the third day after tumor cell injection, treatment wasinitiated with chemotherapy (50 mg/kg gemcitabine i.p., respectively 50mg/kg gemcitabine plus 5 mg/kg oxaliplatin i.p.) and were continuedweekly for six weeks. 24 h after injection of chemotherapeutic agents,800 μg IMAB362 or controls were applied intravenous into the tail vein.IMAB362 treatment was continued weekly until mice were sacrificed. FIG.41A shows growth curves of subcutaneous BxPC3-LVT xenografts. The sizeof s.c. tumors was measured twice weekly (mean+SEM). FIG. 41B showsKaplan-Meier survival curves. Mice were sacrificed when tumors reached avolume of 1400 mm3 or tumors became ulcerous.

FIGS. 42A and 42B show enhancement of antitumoral efficacy bycombination of gemcitabine regimen with IMAB362. BxPC3-LVT xenografttumors were inoculated by injection of 8.5e6 BxPC3-LVT cellssubcutaneous into the flanks of 10 female Hsd:Athymic Nude-Foxnlnu micefor each treatment group. On the third day after tumor cell injection,treatments were initiated with chemotherapy (100 mg/kg gemcitabine i.p.,or 100 mg/kg gemcitabine plus 5 mg/kg oxaliplatin i.p.) and werecontinued weekly for six weeks. 24 h after injection of chemotherapeuticagents, 200 μg (½ dose) or 400 μg (full dose) IMAB362 were appliedintravenous into the tail vein. IMAB362 treatment was continuedsemi-weekly with i.p. and i.v. injections alternating until mice weresacrificed. FIG. 42A shows growth curves of subcutaneous BxPC3-LVTxenografts. The size of s.c. tumors was measured twice weekly(mean+SEM). FIG. 42B shows Kaplan-Meier survival curves. Mice weresacrificed when tumors reached a volume of 1400 mm³ or tumors becameulcerous.

FIGS. 43A and 43B show effect of IMAB362 treatment combined withgemcitabine on MiaPaCa2-LVT xenografts. MiaPaCa2-LVT xenograft tumorswere inoculated by injection of 5e6 MiaPaCa2-LVT cells subcutaneous intothe flank of 10 female Hsd:Athymic Nude-Foxn1nu mice for each treatmentgroup. 4 days after tumor cell injection, treatment was initiated withchemotherapy (50 mg/kg gemcitabine i.p) and were continued weekly forsix weeks. 24 h after injection of chemotherapeutic agents, 200 μgIMAB362 or controls were applied intravenous into the tail vein. IMAB362treatment was continued semi-weekly with i.p. and i.v. injectionsalternating until mice were sacrificed. FIG. 43A shows growth ofsubcutaneous xenografts. The size of tumors was measured twice weekly(mean+SEM). FIG. 43B shows Kaplan-Meier survival curves. Mice weresacrificed when tumors reached a volume of 1400 mm³ or tumors becameulcerous.

FIGS. 44A and 44B show effect of IMAB362 treatment combined withgemcitabine on established MiaPaCa2-LVT xenograft tumors. MiaPaCa2-LVTxenograft tumors were inoculated by injection of 1e7 MiaPaCa2-LVT cellssubcutaneous into the flank of female Hsd:Athymic Nude-Foxn1^(nu) mice.9 days after subcutaneous tumor inoculation, tumor bearing micereorganised in homogenous treatment groups with 8 animals per group andtreatment was initiated. Mice were treated with 150 mg/kg gemcitabinesemi-weekly for 4 weeks i.p. 24 h after gemcitabine injection, 200 μgIMAB362 or controls were applied intravenous into the tail vein.Treatment with 200 μg IMAB362 was continued semi-weekly with i.p. andi.v. injections alternating until mice were sacrificed. In FIG. 44A, thesize of subcutaneous tumors was measured twice weekly (mean+SEM;**=p<0.01). FIG. 44B shows Kaplan-Meier survival curves. Mice weresacrificed when tumor reached a volume of 1400 mm³ or tumor becameulcerous (Log-rank (Mantel-Cox) test; **=p<0.01).

FIGS. 45A, 45B, 45C and 45D show effect of IMAB362 in combination withgemcitabine on lung metastases in Patu8988S xenograft model. 2×10⁶Patu8988S tumor cells were injected intravenously into the tail vein of12 female Hsd:Athymic Nude-Foxn1^(nu) mice per treatment group. Twoweeks after intraveneous tumor cell injection treatment was initiatedwith maintenance treatment of 200 μg IMAB362 semi-weekly (i.v./i.p.)combined with administration of 100 mg/kg gemcitabine i.p. semi-weeklyfor 4 weeks. Control group was treated with 200 μg isotype controlantibody combined with 100 mg/kg gemcitabine semi-weekly. Animals weresacrificed on day 70 post graft. FIG. 45A shows a quantitative PCRanalysis (mean of 3 reactions per sample) of human DNA in lung samplesof IMAB362 and isotype antibody treated mice. Significant difference(P=0.0035, Mann Whitney test) versus isotype control. In FIG. 45B, thepercentage of stained human cells covering the mouse lung surface wasdetermined by computer-based analysis. Immunohistological staining wasperformed with anti human MHC-I antibody (clone EPR1394Y) on paraffinembedded lung tissues (Mean±SEM; P=0.0003, Mann Whitney test). FIGS. 45Cand 45D show examples for immunohistological stainings with anti MHC-Iantibody on Patu8988s lung metastases in IMAB362+gemcitabine (45C) orisotype antibody+gemcitabine treated mice (45D).

DETAILED DESCRIPTION OF THE INVENTION

Although the present invention is described in detail below, it is to beunderstood that this invention is not limited to the particularmethodologies, protocols and reagents described herein as these mayvary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention which will belimited only by the appended claims. Unless defined otherwise, alltechnical and scientific terms used herein have the same meanings ascommonly understood by one of ordinary skill in the art.

In the following, the elements of the present invention will bedescribed. These elements are listed with specific embodiments, however,it should be understood that they may be combined in any manner and inany number to create additional embodiments. The variously describedexamples and preferred embodiments should not be construed to limit thepresent invention to only the explicitly described embodiments. Thisdescription should be understood to support and encompass embodimentswhich combine the explicitly described embodiments with any number ofthe disclosed and/or preferred elements. Furthermore, any permutationsand combinations of all described elements in this application should beconsidered disclosed by the description of the present applicationunless the context indicates otherwise.

Preferably, the terms used herein are defined as described in “Amultilingual glossary of biotechnological terms: (IUPACRecommendations)”, H. G. W. Leuenberger, B. Nagel, and H. Kölbl, Eds.,Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, biochemistry, cellbiology, immunology, and recombinant DNA techniques which are explainedin the literature in the field (cf., e.g., Molecular Cloning: ALaboratory Manual, 2^(nd) Edition, J. Sambrook et al. eds., Cold SpringHarbor Laboratory Press, Cold Spring Harbor 1989).

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated member, integer or step or group of members, integers orsteps but not the exclusion of any other member, integer or step orgroup of members, integers or steps although in some embodiments suchother member, integer or step or group of members, integers or steps maybe excluded, i.e. the subject-matter consists in the inclusion of astated member, integer or step or group of members, integers or steps.The terms “a” and “an” and “the” and similar reference used in thecontext of describing the invention (especially in the context of theclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”), provided herein isintended merely to better illustrate the invention and does not pose alimitation on the scope of the invention otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the invention.

Several documents are cited throughout the text of this specification.Each of the documents cited herein (including all patents, patentapplications, scientific publications, manufacturer's specifications,instructions, etc.), whether supra or infra, are hereby incorporated byreference in their entirety. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention.

The term “CLDN18” relates to claudin 18 and includes any variants,including claudin 18 splice variant 1 (claudin 18.1 (CLDN18.1)) andclaudin 18 splice variant 2 (claudin 18.2 (CLDN18.2)).

The term “CLDN18.2” preferably relates to human CLDN18.2, and, inparticular, to a protein comprising, preferably consisting of the aminoacid sequence according to SEQ ID NO: 1 of the sequence listing or avariant of said amino acid sequence.

The term “CLDN18.1” preferably relates to human CLDN18.1, and, inparticular, to a protein comprising, preferably consisting of the aminoacid sequence according to SEQ ID NO: 2 of the sequence listing or avariant of said amino acid sequence.

The term “variant” according to the invention refers, in particular, tomutants, splice variants, conformations, isoforms, allelic variants,species variants and species homologs, in particular those which arenaturally present. An allelic variant relates to an alteration in thenormal sequence of a gene, the significance of which is often unclear.Complete gene sequencing often identifies numerous allelic variants fora given gene. A species homolog is a nucleic acid or amino acid sequencewith a different species of origin from that of a given nucleic acid oramino acid sequence. The term “variant” shall encompass anyposttranslationally modified variants and conformation variants.

According to the invention, the term “CLDN18.2 positive cancer” means acancer involving cancer cells expressing CLDN18.2, preferably on thesurface of said cancer cells.

“Cell surface” is used in accordance with its normal meaning in the art,and thus includes the outside of the cell which is accessible to bindingby proteins and other molecules. For example, a transmembrane proteinhaving one or more extracellular portions is considered as beingexpressed on the cell surface.

CLDN18.2 is expressed on the surface of cells if it is located at thesurface of said cells and is accessible to binding by CLDN18.2-specificantibodies added to the cells.

According to the invention, CLDN18.2 is not substantially expressed in acell if the level of expression is lower compared to expression instomach cells or stomach tissue. Preferably, the level of expression isless than 10%, preferably less than 5%, 3%, 2%, 1%, 0.5%, 0.1% or 0.05%of the expression in stomach cells or stomach tissue or even lower.Preferably, CLDN18.2 is not substantially expressed in a cell if thelevel of expression exceeds the level of expression in non-canceroustissue other than stomach by no more than 2-fold, preferably 1,5-fold,and preferably does not exceed the level of expression in saidnon-cancerous tissue. Preferably, CLDN18.2 is not substantiallyexpressed in a cell if the level of expression is below the detectionlimit and/or if the level of expression is too low to allow binding byCLDN18.2-specific antibodies added to the cells.

According to the invention, CLDN18.2 is expressed in a cell if the levelof expression exceeds the level of expression in non-cancerous tissueother than stomach preferably by more than 2-fold, preferably 10-fold,100-fold, 1000-fold, or 10000-fold. Preferably, CLDN18.2 is expressed ina cell if the level of expression is above the detection limit and/or ifthe level of expression is high enough to allow binding byCLDN18.2-specific antibodies added to the cells. Preferably, CLDN18.2expressed in a cell is expressed or exposed on the surface of said cell.

According to the invention, the term “disease” refers to anypathological state, including cancer, in particular those forms ofcancer described herein. Any reference herein to cancer or particularforms of cancer also includes cancer metastasis thereof. In a preferredembodiment, a disease to be treated according to the present applicationinvolves cells expressing CLDN18.2.

“Diseases associated with cells expressing CLDN18.2” or similarexpressions means according to the invention that CLDN18.2 is expressedin cells of a diseased tissue or organ. In one embodiment, expression ofCLDN18.2 in cells of a diseased tissue or organ is increased compared tothe state in a healthy tissue or organ. An increase refers to anincrease by at least 10%, in particular at least 20%, at least 50%, atleast 100%, at least 200%, at least 500%, at least 1000%, at least10000% or even more. In one embodiment, expression is only found in adiseased tissue, while expression in a corresponding healthy tissue isrepressed. For example, CLDN18.2 is expressed in pancreatic cancertissue while expression is not detectable in non-cancerous pancreatictissue. According to the invention, diseases associated with cellsexpressing CLDN18.2 include cancer diseases. Furthermore, according tothe invention, cancer diseases preferably are those wherein the cancercells express CLDN18.2.

As used herein, a “cancer disease” or “cancer” includes a diseasecharacterized by aberrantly regulated cellular growth, proliferation,differentiation, adhesion, and/or migration. By “cancer cell” is meantan abnormal cell that grows by a rapid, uncontrolled cellularproliferation and continues to grow after the stimuli that initiated thenew growth cease. Preferably, a “cancer disease” is characterized bycells expressing CLDN18.2 and a cancer cell expresses CLDN18.2.

A cell expressing CLDN18.2 preferably is a cancer cell, preferably ofthe cancers described herein.

According to the invention, a “carcinoma” is a malignant tumor derivedfrom epithelial cells.

“Adenocarcinoma” is a cancer that originates in glandular tissue. Thistissue is also part of a larger tissue category known as epithelialtissue. Epithelial tissue includes skin, glands and a variety of othertissue that lines the cavities and organs of the body. Epithelium isderived embryologically from ectoderm, endoderm and mesoderm. To beclassified as adenocarcinoma, the cells do not necessarily need to bepart of a gland, as long as they have secretory properties. This form ofcarcinoma can occur in some higher mammals, including humans. Welldifferentiated adenocarcinomas tend to resemble the glandular tissuethat they are derived from, while poorly differentiated may not. Bystaining the cells from a biopsy, a pathologist will determine whetherthe tumor is an adenocarcinoma or some other type of cancer.Adenocarcinomas can arise in many tissues of the body due to theubiquitous nature of glands within the body. While each gland may not besecreting the same substance, as long as there is an exocrine functionto the cell, it is considered glandular and its malignant form istherefore named adenocarcinoma. Malignant adenocarcinomas invade othertissues and often metastasize given enough time to do so.

The pancreas, an organ of endodermal derivation, is the key regulator ofprotein and carbohydrate digestion and glucose homeostasis. The exocrinepancreas (80% of the tissue mass of the organ) is composed of abranching network of acinar and duct cells that produce and deliverdigestive enzymes into the gastrointestinal tract. The acinar cells,which are organized in functional units along the duct network,synthesize and secrete enzymes into the ductal lumen in response to cuesfrom the stomach and duodenum. Within the acinar units near the ductsare centroacinar cells. The endocrine pancreas, which regulatesmetabolism and glucose homeostasis through the secretion of hormonesinto the bloodstream, is composed of four specialized endocrine celltypes gathered together into clusters called Islets of Langerhans.

Pancreatic cancer is a malignant neoplasm originating from transformedcells arising in tissues forming the pancreas. Pancreatic cancer is thefourth most common cause of cancer-related deaths in the United Statesand the eighth worldwide. Early pancreatic cancer often does not causesymptoms, and the later symptoms are usually nonspecific and varied.Therefore, pancreatic cancer is often not diagnosed until it isadvanced. Pancreatic cancer has a poor prognosis: for all stagescombined, the 1- and 5-year relative survival rates are 25% and 6%,respectively. For local disease the 5-year survival is approximately 20%while the median survival for locally advanced and for metastaticdisease, which collectively represent over 80% of individuals, is about10 and 6 months respectively.

Pancreatic cancer includes adenocarcinomas (tumors exhibiting glandulararchitecture) arising within the exocrine component of the pancreas andneuroendocrine carcinomas arising from islet cells.

The most common form of pancreatic cancer, ductal adenocarcinoma, istypically characterized by moderately to poorly differentiated glandularstructures on microscopic examination. Pancreatic ductal adenocarcinoma(PDAC) commonly arises in the head of the pancreas with infiltrationinto surrounding tissues including lymphatics, spleen, and peritonealcavity, and with metastasis to the liver and lungs. PDAC primarilyexhibits a glandular pattern with duct-like structures and varyingdegrees of cellular atypia and differentiation. Less common subtypes ofPDAC include colloid, adenosquamous, or sarcomatoid histology. Oftenwithin an individual tumor, there are regional differences in histology,tumor grade, and degree of differentiation. Even the smallest primarylesions commonly exhibit perineural and lympho-vascular invasion,suggesting a propensity for early distant spread.

The second most common type of exocrine pancreas cancer is mucinous.Mucinous adenocarcinoma produces a large volume of mucin that results ina cystic appearance on imaging studies.

Pancreatic neuroendocrine tumors form in hormone-making cells (isletcells) of the pancreas. Acinic cell neoplasms arise from the acinarcells of the pancreas.

According to the invention, the term “cancer” also includes cancermetastasis of a primary tumor such as primary pancreatic cancer. Thus,if reference is made, for example, to pancreatic cancer, this alsoincludes metastasis of the pancreatic cancer, for example metastasis tothe lung, liver and/or lymph nodes.

By “metastasis” is meant the spread of cancer cells from its originalsite to another part of the body. The formation of metastasis is a verycomplex process and depends on detachment of malignant cells from theprimary tumor, invasion of the extracellular matrix, penetration of theendothelial basement membranes to enter the body cavity and vessels, andthen, after being transported by the blood, infiltration of targetorgans. Finally, the growth of a new tumor at the target site depends onangiogenesis. Tumor metastasis often occurs even after the removal ofthe primary tumor because tumor cells or components may remain anddevelop metastatic potential. In one embodiment, the term “metastasis”according to the invention relates to “distant metastasis” which relatesto a metastasis which is remote from the primary tumor and the regionallymph node system. In one embodiment, the term “metastasis” according tothe invention relates to lymph node metastasis. One particular form ofmetastasis which is treatable using the therapy of the invention ismetastasis originating from pancreatic cancer as primary site. Inpreferred embodiments such pancreatic cancer metastasis is metastasisinto lymph nodes, metastasis into lung and/or metastasis into liver.

Krukenberg tumor is an uncommon metastatic tumor of the ovary accountingfor 1% to 2% of all ovarian tumors. Krukenberg tumor is a metastaticsignet ring cell adenocarcinoma of the ovary. Stomach is the primarysite in most Krukenberg tumor cases (70%). Carcinomas of colon,appendix, and breast (mainly invasive lobular carcinoma) are the nextmost common primary sites. Rare cases of Krukenberg tumor originatingfrom carcinomas of the gallbladder, biliary tract, pancreas, smallintestine, ampulla of Vater, cervix, and urinary bladder/urachus havebeen reported.

A refractory cancer is a malignancy for which a particular treatment isineffective, which is either initially unresponsive to treatment, orwhich becomes unresponsive over time.

By “treat” is meant to administer a compound or composition or acombination of compounds or compositions to a subject in order toprevent or eliminate a disease, including reducing the size of a tumoror the number of tumors in a subject; arrest or slow a disease in asubject; inhibit or slow the development of a new disease in a subject;decrease the frequency or severity of symptoms and/or recurrences in asubject who currently has or who previously has had a disease; and/orprolong, i.e. increase the lifespan of the subject.

In particular, the term “treatment of a disease” includes curing,shortening the duration, ameliorating, preventing, slowing down orinhibiting progression or worsening, or preventing or delaying the onsetof a disease or the symptoms thereof.

The term “patient” means according to the invention a subject fortreatment, in particular a diseased subject, including human beings,nonhuman primates or another animals, in particular mammals such ascows, horses, pigs, sheeps, goats, dogs, cats or rodents such as miceand rats. In a particularly preferred embodiment, a patient is a humanbeing.

The term “agent stabilizing or increasing expression of CLDN18.2” refersto an agent or a combination of agents the provision of which to cellsresults in increased RNA and/or protein levels of CLDN18.2 in saidcells, preferably in increased levels of CLDN18.2 protein on the cellsurface, compared to the situation where the cells are not provided withthe agent or the combination of agents. Preferably, the cells are cancercells, in particular cancer cells expressing CLDN18.2 and thus are atarget for CLDN18.2 binding antibodies, such as cells of the cancertypes described herein, in particular pancreatic cancer. The term “agentstabilizing or increasing expression of CLDN18.2” refers, in particular,to an agent or a combination of agents the provision of which to cellsresults in a higher density of CLDN18.2 on the surface of said cellscompared to the situation where the cells are not provided with theagent or the combination of agents. “Stabilizing expression of CLDN18.2”includes, in particular, the situation where the agent or thecombination of agents prevents a decrease or reduces a decrease inexpression of CLDN18.2, e.g. expression of CLDN18.2 would decreasewithout provision of the agent or the combination of agents andprovision of the agent or the combination of agents prevents saiddecrease or reduces said decrease of CLDN18.2 expression. “Increasingexpression of CLDN18.2” includes, in particular, the situation where theagent or the combination of agents increases expression of CLDN18.2,e.g. expression of CLDN18.2 would decrease, remain essentially constantor increase without provision of the agent or the combination of agentsand provision of the agent or the combination of agents increasesCLDN18.2 expression compared to the situation without provision of theagent or the combination of agents so that the resulting expression ishigher compared to the situation where expression of CLDN18.2 woulddecrease, remain essentially constant or increase without provision ofthe agent or the combination of agents.

According to the invention, the term “agent stabilizing or increasingexpression of CLDN18.2” includes chemotherapeutic agents or combinationsof chemotherapeutic agents such as cytostatic agents. Chemotherapeuticagents may affect cells in one of the following ways: (1) damage the DNAof the cells so they can no longer reproduce, (2) inhibit the synthesisof new DNA strands so that no cell replication is possible, (3) stop themitotic processes of the cells so that the cells cannot divide into twocells.

According to the invention, the term “agent stabilizing or increasingexpression of CLDN18.2” preferably relates to an agent or a combinationof agents such a cytostatic compound or a combination of cytostaticcompounds the provision of which to cells, in particular cancer cells,results in the cells being arrested in or accumulating in one or morephases of the cell cycle, preferably in one or more phases of the cellcycle other than the G1- and G0-phases, preferably other than theG1-phase, preferably in one or more of the G2- or S-phase of the cellcycle such as the G1/G2-, S/G2-, G2- or S-phase of the cell cycle. Theterm “cells being arrested in or accumulating in one or more phases ofthe cell cycle” means that the percentage of cells which are in said oneor more phases of the cell cycle increases. Each cell goes through acycle comprising four phases in order to replicate itself. The firstphase called G1 is when the cell prepares to replicate its chromosomes.The second stage is called S, and in this phase DNA synthesis occurs andthe DNA is duplicated. The next phase is the G2 phase, when the RNA andprotein duplicate. The final stage is the M stage, which is the stage ofactual cell division. In this final stage, the duplicated DNA and RNAsplit and move to separate ends of the cell, and the cell actuallydivides into two identical, functional cells. Chemotherapeutic agentswhich are DNA damaging agents usually result in an accumulation of cellsin the G1 and/or G2 phase. Chemotherapeutic agents which block cellgrowth by interfering with DNA synthesis such as antimetabolites usuallyresult in an accumulation of cells in the S-phase. Examples of thesedrugs are gemcitabine, 6-mercaptopurine and 5-fluorouracil.

According to the invention, the term “agent stabilizing or increasingexpression of CLDN18.2” includes nucleoside analogs such as gemcitabine,5-fluorouracil or prodrugs thereof, platinum compounds such asoxaliplatin and cisplatin, taxanes such as paclitaxel and docetaxel, andcamptothecin analogs such as irinotecan and topotecan, and combinationsof drugs such as combinations of drugs comprising one or more ofgemcitabine, oxaliplatin and 5-fluorouracil such as a combination ofdrugs comprising gemcitabine and oxaliplatin, gemcitabine and5-fluorouracil, oxaliplatin and 5-fluorouracil or other drugcombinations described herein. According to the invention a reference toan agent stabilizing or increasing expression of CLDN18.2, such as areference to a nucleoside analog, a platinum compound, a camptothecinanalog or a taxane, for example, a reference to gemcitabine,5-fluorouracil, oxaliplatin, irinotecan or paclitaxel is to include anyprodrug such as ester, salt or derivative such as conjugate of saidagent. Examples are conjugates of said agent with a carrier substance,e.g. protein-bound paclitaxel such as albumin-bound paclitaxel.Preferably, salts of said agent are pharmaceutically acceptable.

In one preferred embodiment, an “agent stabilizing or increasingexpression of CLDN18.2” is or comprises an “agent inducing immunogeniccell death”.

In specific circumstances, cancer cells can enter a lethal stresspathway linked to the emission of a spatiotemporally defined combinationof signals that is decoded by the immune system to activatetumor-specific immune responses (Zitvogel L. et al. (2010) Cell 140:798-804). In such scenario cancer cells are triggered to emit signalsthat are sensed by innate immune effectors such as dendritic cells totrigger a cognate immune response that involves CD8+ T cells and IFN-γsignalling so that tumor cell death may elicit a productive anticancerimmune response. These signals include the pre-apoptotic exposure of theendoplasmic reticulum (ER) chaperon calreticulin (CRT) at the cellsurface, the pre-apoptotic secretion of ATP, and the post-apoptoticrelease of the nuclear protein HMGB1. Together, these processesconstitute the molecular determinants of immunogenic cell death (ICD).Anthracyclines, oxaliplatin, and γ irradiation are able to induce allsignals that define ICD, while cisplatin, for example, which isdeficient in inducing CRT translocation from the ER to the surface ofdying cells—a process requiring ER stress—requires complementation bythapsigargin, an ER stress inducer.

According to the invention, the term “agent inducing immunogenic celldeath” refers to an agent or a combination of agents which when providedto cells, in particular cancer cells, is capable of inducing the cellsto enter a lethal stress pathway which finally results in tumor-specificimmune responses. In particular, an agent inducing immunogenic celldeath when provided to cells induces the cells to emit aspatiotemporally defined combination of signals, including, inparticular, the pre-apoptotic exposure of the endoplasmic reticulum (ER)chaperon calreticulin (CRT) at the cell surface, the pre-apoptoticsecretion of ATP, and the post-apoptotic release of the nuclear proteinHMGB1.

According to the invention, the term “agent inducing immunogenic celldeath” includes anthracyclines and oxaliplatin.

The term “nucleoside analog” refers to a structural analog of anucleoside, a category that includes both purine analogs and pyrimidineanalogs.

The term “gemcitabine” is a compound which is a nucleoside analog of thefollowing formula:

In particular, the term refers to the compound4-amino-1-(2-deoxy-2,2-difluoro-β-D-erythro-pentofuranosyl)pyrimidin-2(1H)-oneor4-amino-1-[(2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]-1,2-dihydropyrimidin-2-one.

According to the invention, gemcitabine is preferably administered bythe intravenous route. Preferably, gemcitabine is administered in doseranges of 0.5 to 2 g/m², preferably 0.8 to 1.5 g/m², more preferably 1to 1.2 g/m² of body surface area. For example, gemcitabine may be givenat a dose of 1000 mg per square meter weekly for 7 of 8 weeks and thenweekly for 3 of 4 weeks.

The term “nucleoside analog” includes fluoropyrimidine derivatives suchas fluorouracil and prodrugs thereof. The term “fluorouracil” or“5-fluorouracil” (5-FU or f5U) (sold under the brand names Adrucil,Carac, Efudix, Efudex and Fluoroplex) is a compound which is apyrimidine analog of the following formula:

In particular, the term refers to the compound5-fluoro-1H-pyrimidine-2,4-dione.

The term “capecitabine” (Xeloda, Roche) refers to a chemotherapeuticagent that is a prodrug that is converted into 5-FU in the tissues.Capecitabine which may be orally administered has the following formula:

In particular, the term refers to the compound pentyl[1-(3,4-dihydroxy-5-methyltetrahydrofuran-2-yl)-5-fluoro-2-oxo-1H-pyrimidin-4-yl]carbamate.

According to the invention, the term “platinum compound” refers tocompounds containing platinum in their structure such as platinumcomplexes and includes compounds such as cisplatin, carboplatin andoxaliplatin.

The term “cisplatin” or “cisplatinum” refers to the compoundcis-diamminedichloroplatinum(II) (CDDP) of the following formula:

The term “carboplatin” refers to the compoundcis-diammine(1,1-cyclobutanedicarboxylato)platinum(II) of the followingformula:

The term “oxaliplatin” refers to a compound which is a platinum compoundthat is complexed to a diaminocyclohexane carrier ligand of thefollowing formula:

In particular, the term “oxaliplatin” refers to the compound[(1R,2R)-cyclohexane-1,2-diamine](ethanedioato-O,O′)platinum(II).Oxaliplatin for injection is also marketed under the trade nameEloxatine.

Taxanes are a class of diterpene compounds that were first derived fromnatural sources such as plants of the genus Taxus, but some have beensynthesized artificially. The principal mechanism of action of thetaxane class of drugs is the disruption of microtubule function, therebyinhibiting the process of cell division. Taxanes include docetaxel(Taxotere) and paclitaxel (Taxol).

According to the invention, the term “docetaxel” refers to a compoundhaving the following formula:

In particular, the term “docetaxel” refers to the compound1,7β,10β-trihydroxy-9-oxo-5β,20-epoxytax-11-ene-2α,4,13α-triyl 4-acetate2-benzoate13-{(2R,3S)-3-[(tert-butoxycarbonyl)-amino]-2-hydroxy-3-phenylpropanoate}.

According to the invention, the term “paclitaxel” refers to a compoundhaving the following formula:

In particular, the term “paclitaxel” refers to the compound(2α,4α,5β,7β,10β,13α)-4,10-bis-(acetyloxy)-13-{[(2R,3S)-3-(benzoylamino)-2-hydroxy-3-phenylpropanoyl]oxy}-1,7-dihydroxy-9-oxo-5,20-epoxytax-11-en-2-ylbenzoate.

According to the invention, the term “camptothecin analog” refers toderivatives of the compound camptothecin (CPT;(S)-4-ethyl-4-hydroxy-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14-(4H,12H)-dione).Preferably, the term “camptothecin analog” refers to compoundscomprising the following structure:

According to the invention, preferred camptothecin analogs areinhibitors of DNA enzyme topoisomerase I (topo I). Preferredcamptothecin analogs according to the invention are irinotecan andtopotecan.

Irinotecan is a drug preventing DNA from unwinding by inhibition oftopoisomerase I. In chemical terms, it is a semisynthetic analogue ofthe natural alkaloid camptothecin having the following formula:

In particular, the term “irinotecan” refers to the compound(S)-4,11-diethyl-3,4,12,14-tetrahydro-4-hydroxy-3,14-dioxo1H-pyrano[3′,4′:6,7]-indolizino[1,2-b]quinolin-9-yl-[1,4′-bipiperidine]-1′-carboxylate.

Topotecan is a topoisomerase inhibitor of the formula:

In particular, the term “topotecan” refers to the compound(S)-10-[(dimethylamino)methyl]-4-ethyl-4,9-dihydroxy-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H,12H)-dionemonohydrochloride.

Anthracyclines are a class of drugs commonly used in cancer chemotherapythat are also antibiotics. Structurally, all anthracyclines share acommon four-ringed 7,8,9,10-tetrahydrotetracene-5,12-quinone structureand usually require glycosylation at specific sites.

Anthracyclines preferably bring about one or more of the followingmechanisms of action: 1. Inhibiting DNA and RNA synthesis byintercalating between base pairs of the DNA/RNA strand, thus preventingthe replication of rapidly-growing cancer cells. 2. Inhibitingtopoisomerase II enzyme, preventing the relaxing of supercoiled DNA andthus blocking DNA transcription and replication. 3. Creatingiron-mediated free oxygen radicals that damage the DNA and cellmembranes.

According to the invention, the term “anthracycline” preferably relatesto an agent, preferably an anticancer agent for inducing apoptosis,preferably by inhibiting the rebinding of DNA in topoisomerase II.

Preferably, according to the invention, the term “anthracycline”generally refers to a class of compounds having the following ringstructure

including analogs and derivatives, pharmaceutical salts, hydrates,esters, conjugates and prodrugs thereof.

Examples of anthracyclines and anthracycline analogs include, but arenot limited to, daunorubicin (daunomycin), doxorubicin (adriamycin),epirubicin, idarubicin, rhodomycin, pyrarubicin, valrubicin,N-trifluoro-acetyl doxorubicin-14-valerate, aclacinomycin,morpholinodoxorubicin (morpholino-DOX), cyanomorpholino-doxorubicin(cyano-morpholino-DOX), 2-pyrrolino-doxorubicin (2-PDOX),5-iminodaunomycin, mitoxantrone and aclacinomycin A (aclarubicin).Mitoxantrone is a member of the anthracendione class of compounds, whichare anthracycline analogs that lack the sugar moiety of theanthracyclines but retain the planar polycylic aromatic ring structurethat permits intercalation into DNA.

Particularly preferred as anthracyline according to the invention is acompound of the following formula:

wherein

R₁ is selected from the group consisting of H and OH, R₂ is selectedfrom the group consisting of H and OMe, R₃ is selected from the groupconsisting of H and OH, and R₄ is selected from the group consisting ofH and OH.

In one embodiment, R₁ is H, R₂ is OMe, R₃ is H, and R₄ is OH. In anotherembodiment, R₁ is OH, R₂ is OMe, R₃ is H, and R₄ is OH. In anotherembodiment, R₁ is OH, R₂ is OMe, R₃ is OH, and R₄ is H. In anotherembodiment, R₁ is H, R₂ is H, R₃ is H, and R₄ is OH.

Specifically contemplated as anthracycline in the context of the presentinvention is epirubicin. Epirubicin is an anthracycline drug which hasthe following formula:

and is marketed under the trade name Ellence in the US and Pharmorubicinor Epirubicin Ebewe elsewhere. In particular, the term “epirubicin”refers to the compound(8R,10S)-10-[(2S,4S,5R,6S)-4-amino-5-hydroxy-6-methyl-oxan-2-yl]oxy-6,11-dihydroxy-8-(2-hydroxyacetyl)-1-methoxy-8-methyl-9,10-dihydro-7H-tetracen-5,12-dion.Epirubicin is favoured over doxorubicin, the most popular anthracycline,in some chemotherapy regimens as it appears to cause fewer side-effects.

According to the invention, an agent stabilizing or increasingexpression of CLDN18.2 may be a chemotherapeutic agent, in particular achemotherapeutic agent established in cancer treatment and may be partof a combination of drugs such as a combination of drugs established foruse in cancer treatment. Such combination of drugs may be a drugcombination used in chemotherapy, and may be a drug combination as usedin the FOLFIRINOX chemotherapeutic regimen.

The drug combination used in FOLFIRINOX chemotherapy comprises ofleucovorin, fluorouracil, irinotecan (such as irinotecan hydrochloride)and oxaliplatin. Oxaliplatin may be given at 85 mg per square meter ofbody-surface area; irinotecan at 180 mg per square meter; leucovorin at400 mg per square meter; and fluorouracil at 400 mg per square metergiven as a bolus followed by 5-fluorouracil at 2400 mg per square metergiven as a continuous infusion of preferably 46-hours, preferably every2 weeks).

The term “folinic acid” or “leucovorin” refers to a compound useful insynergistic combination with the chemotherapy agent 5-fluorouracil.Thus, if reference is made herein to the administration of5-fluorouracil or a prodrug thereof, said administration in oneembodiment may comprise an administration in conjunction with folinicacid. Folinic acid has the following formula:

In particular, the term refers to the compound(2S)-2-{[4-[(2-amino-5-formyl-4-oxo-5,6,7,8-tetrahydro-1H-pteridin-6-yl)methylamino]benzoyl]amino}pentanedioicacid.

γδ T cells (gamma delta T cells) represent a small subset of T cellsthat possess a distinct T cell receptor (TCR) on their surface. Amajority of T cells have a TCR composed of two glycoprotein chainscalled α- and β-TCR chains. In contrast, in γδ T cells, the TCR is madeup of one γ-chain and one δ-chain. This group of T cells is usually muchless common than αβ T cells. Human γδ T cells play an important role instress-surveillance responses like infectious diseases and autoimmunity.Transformation-induced changes in tumors are also suggested to causestress-surveillance responses mediated by γδ T cells and enhanceantitumor immunity. Importantly, after antigen engagement, activated γδT cells at lesional sites provide cytokines (e.g. INFγ, TNFα) and/orchemokines mediating recruitment of other effector cells and showimmediate effector functions such as cytotoxicity (via death receptorand cytolytic granules pathways) and ADCC.

The majority of γδ T cells in peripheral blood express the Vγ9Vδ2 T cellreceptor (TCRγδ). Vγ9Vδ2 T cells are unique to humans and primates andare assumed to play an early and essential role in sensing “danger” byinvading pathogens as they expand dramatically in many acute infectionsand may exceed all other lymphocytes within a few days, e.g. intuberculosis, salmonellosis, ehrlichiosis, brucellosis, tularemia,listeriosis, toxoplasmosis, and malaria.

γδ T cells respond to small non-peptidic phosphorylated antigens(phosphoantigens) such as pyrophosphates synthesized in bacteria andisopentenyl pyrophosphate (IPP) produced in mammalian cells through themevalonate pathway. Whereas IPP production in normal cells is notsufficient for activation of γδ T cells, dysregulation of the mevalonatepathway in tumor cells leads to accumulation of IPP and γδ T cellactivation. IPPs can also be therapeutically increased byaminobisphosphonates, which inhibit the mevalonate pathway enzymefarnesyl pyrophosphate synthase (FPPS). Among others, zoledronic acid(ZA, zoledronate, Zometa™, Novartis) represents such anaminobiphosphonate, which is already clinically administered to patientsfor the treatment of osteoporosis and metastasic bone disease. Upontreatment of PBMCs in vitro, ZA is taken up especially by monocytes. IPPaccumulates in the monocytes and they differentiate toantigen-presenting cells stimulating development of γδ T cells. In thissetting, the addition of interleukin-2 (IL-2) is preferred as growth andsurvival factor for activated γδ T cells. Finally, certain alkylatedamines have been described to activate Vγ9Vδ2 T cells in vitro, howeveronly at millimolar concentrations.

According to the invention, the term “agent stimulating γδ T cells”relates to compounds stimulating development of γδ T cells, inparticular Vγ9Vδ2 T cells, in vitro and/or in vivo, in particular byinducing activation and expansion of γδ T cells. Preferably, the termrelates to compounds which in vitro and/or in vivo increase isopentenylpyrophosphate (IPP) produced in mammalian cells, preferably byinhibiting the mevalonate pathway enzyme farnesyl pyrophosphate synthase(FPPS).

One particular group of compounds stimulating γδ T cells arebisphosphonates, in particular nitrogen-containing bisphosphonates(N-bisphosphonates; aminobisphosphonates).

For example, suitable bisphosphonates for use in the invention mayinclude one or more of the following compounds including analogs andderivatives, pharmaceutical salts, hydrates, esters, conjugates andprodrugs thereof:

-   [1-hydroxy-2-(1H-imidazol-1-yl)ethane-1,1-diyl]bis(phosphonic acid),    zoledronic acid, e.g. zoledronate;-   (dichloro-phosphono-methyl)phosphonic acid, e.g. clodronate-   {1-hydroxy-3-[methyl(pentyl)amino]propane-1,1-diyl}bis(phosphonic    acid), ibandronic acid, e.g. ibandronate-   (3-amino-1-hydroxypropane-1,1-diyl)bis(phosphonic acid), pamidronic    acid, e.g. pamidronate;-   (1-hydroxy-1-phosphono-2-pyridin-3-yl-ethyl)phosphonic acid,    risedronic acid, e.g. risedronate;-   (1-Hydroxy-2-imidazo[1,2-a]pyridin-3-yl-1-phosphonoethyl)phosphonic    acid, minodronic acid;-   [3-(dimethylamino)-1-hydroxypropane-1,1-diyl]bis(phosphonic acid),    olpadronic acid.-   [4-amino-1-hydroxy-1-(hydroxy-oxido-phosphoryl)-butyl]phosphonic    acid, alendronic acid, e.g. alendronate;-   [(Cycloheptylamino)methylene]bis(phosphonic acid), incadronic acid;-   (1-hydroxyethan-1,1-diyl)bis(phosphonic acid), etidronic acid, e.g.    etidronate; and-   {[(4-chlorophenyl)thio]methylene}bis(phosphonic acid), tiludronic    acid.

According to the invention, zoledronic acid (INN) or zoledronate(marketed by Novartis under the trade names Zometa, Zomera, Aclasta andReclast) is a particularly preferred bisphosphonate. Zometa is used toprevent skeletal fractures in patients with cancers such as multiplemyeloma and prostate cancer, as well as for treating osteoporosis. Itcan also be used to treat hypercalcemia of malignancy and can be helpfulfor treating pain from bone metastases.

In one particularly preferred embodiment, an agent stimulating γδ Tcells according to the invention is administered in combination withIL-2. Such combination has been shown to be particularly effective inmediating expansion and activation of γ9δ2 T cells.

Interleukin-2 (IL-2) is an interleukin, a type of cytokine signalingmolecule in the immune system. It is a protein that attracts lymphocytesand is part of the body's natural response to microbial infection, andin discriminating between foreign (non-self) and self. IL-2 mediates itseffects by binding to IL-2 receptors, which are expressed bylymphocytes.

The IL-2 used according to the invention may be any IL-2 supporting orenabling the stimulation of γδ T cells and may be derived from anyspecies, preferably human. Il-2 may be isolated, recombinantly producedor synthetic IL-2 and may be naturally occurring or modified IL-2.

The term “antigen” relates to an agent such as a protein or peptidecomprising an epitope against which an immune response is directedand/or is to be directed. In a preferred embodiment, an antigen is atumor-associated antigen, such as CLDN18.2, i.e., a constituent ofcancer cells which may be derived from the cytoplasm, the cell surfaceand the cell nucleus, in particular those antigens which are produced,preferably in large quantity, intracellular or as surface antigens oncancer cells.

In the context of the present invention, the term “tumor-associatedantigen” preferably relates to proteins that are under normal conditionsspecifically expressed in a limited number of tissues and/or organs orin specific developmental stages and are expressed or aberrantlyexpressed in one or more tumor or cancer tissues. In the context of thepresent invention, the tumor-associated antigen is preferably associatedwith the cell surface of a cancer cell and is preferably not or onlyrarely expressed in normal tissues.

The term “epitope” refers to an antigenic determinant in a molecule,i.e., to the part in a molecule that is recognized by the immune system,for example, that is recognized by an antibody. For example, epitopesare the discrete, three-dimensional sites on an antigen, which arerecognized by the immune system. Epitopes usually consist of chemicallyactive surface groupings of molecules such as amino acids or sugar sidechains and usually have specific three dimensional structuralcharacteristics, as well as specific charge characteristics.Conformational and non-conformational epitopes are distinguished in thatthe binding to the former but not the latter is lost in the presence ofdenaturing solvents. An epitope of a protein such as CLDN18.2 preferablycomprises a continuous or discontinuous portion of said protein and ispreferably between 5 and 100, preferably between 5 and 50, morepreferably between 8 and 30, most preferably between 10 and 25 aminoacids in length, for example, the epitope may be preferably 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 aminoacids in length.

The term “antibody” refers to a glycoprotein comprising at least twoheavy (H) chains and two light (L) chains inter-connected by disulfidebonds, and includes any molecule comprising an antigen binding portionthereof. The term “antibody” includes monoclonal antibodies andfragments or derivatives of antibodies, including, without limitation,human antibodies, humanized antibodies, chimeric antibodies, singlechain antibodies, e.g., scFv's and antigen-binding antibody fragmentssuch as Fab and Fab′ fragments and also includes all recombinant formsof antibodies, e.g., antibodies expressed in prokaryotes, unglycosylatedantibodies, and any antigen-binding antibody fragments and derivativesas described herein. Each heavy chain is comprised of a heavy chainvariable region (abbreviated herein as VH) and a heavy chain constantregion. Each light chain is comprised of a light chain variable region(abbreviated herein as VL) and a light chain constant region. The VH andVL regions can be further subdivided into regions of hypervariability,termed complementarity determining regions (CDR), interspersed withregions that are more conserved, termed framework regions (FR). Each VHand VL is composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and lightchains contain a binding domain that interacts with an antigen. Theconstant regions of the antibodies may mediate the binding of theimmunoglobulin to host tissues or factors, including various cells ofthe immune system (e.g., effector cells) and the first component (C1q)of the classical complement system.

The antibodies described herein may be human antibodies. The term “humanantibody”, as used herein, is intended to include antibodies havingvariable and constant regions derived from human germline immunoglobulinsequences. The human antibodies described herein may include amino acidresidues not encoded by human germline immunoglobulin sequences (e.g.,mutations introduced by random or site-specific mutagenesis in vitro orby somatic mutation in vivo).

The term “humanized antibody” refers to a molecule having an antigenbinding site that is substantially derived from an immunoglobulin from anon-human species, wherein the remaining immunoglobulin structure of themolecule is based upon the structure and/or sequence of a humanimmunoglobulin. The antigen binding site may either comprise completevariable domains fused onto constant domains or only the complementaritydetermining regions (CDR) grafted onto appropriate framework regions inthe variable domains. Antigen binding sites may be wild-type or modifiedby one or more amino acid substitutions, e.g. modified to resemble humanimmunoglobulins more closely. Some forms of humanized antibodiespreserve all CDR sequences (for example a humanized mouse antibody whichcontains all six CDRs from the mouse antibody). Other forms have one ormore CDRs which are altered with respect to the original antibody.

The term “chimeric antibody” refers to those antibodies wherein oneportion of each of the amino acid sequences of heavy and light chains ishomologous to corresponding sequences in antibodies derived from aparticular species or belonging to a particular class, while theremaining segment of the chain is homologous to corresponding sequencesin another. Typically the variable region of both light and heavy chainsmimics the variable regions of antibodies derived from one species ofmammals, while the constant portions are homologous to sequences ofantibodies derived from another. One clear advantage to such chimericforms is that the variable region can conveniently be derived frompresently known sources using readily available B-cells or hybridomasfrom non-human host organisms in combination with constant regionsderived from, for example, human cell preparations. While the variableregion has the advantage of ease of preparation and the specificity isnot affected by the source, the constant region being human, is lesslikely to elicit an immune response from a human subject when theantibodies are injected than would the constant region from a non humansource. However the definition is not limited to this particularexample.

The terms “antigen-binding portion” of an antibody (or simply “bindingportion”) or “antigen-binding fragment” of an antibody (or simply“binding fragment”) or similar terms refer to one or more fragments ofan antibody that retain the ability to specifically bind to an antigen.It has been shown that the antigen-binding function of an antibody canbe performed by fragments of a full-length antibody. Examples of bindingfragments encompassed within the term “antigen-binding portion” of anantibody include (i) Fab fragments, monovalent fragments consisting ofthe VL, VH, CL and CH domains; (ii) F(ab′)₂ fragments, bivalentfragments comprising two Fab fragments linked by a disulfide bridge atthe hinge region; (iii) Fd fragments consisting of the VH and CHdomains; (iv) Fv fragments consisting of the VL and VH domains of asingle arm of an antibody, (v) dAb fragments (Ward et al., (1989) Nature341: 544-546), which consist of a VH domain; (vi) isolatedcomplementarity determining regions (CDR), and (vii) combinations of twoor more isolated CDRs which may optionally be joined by a syntheticlinker. Furthermore, although the two domains of the Fv fragment, VL andVH, are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the VL and VH regions pair to formmonovalent molecules (known as single chain Fv (scFv); see e.g., Bird etal. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl.Acad. Sci. USA 85: 5879-5883). Such single chain antibodies are alsointended to be encompassed within the term “antigen-binding fragment” ofan antibody. A further example is binding-domain immunoglobulin fusionproteins comprising (i) a binding domain polypeptide that is fused to animmunoglobulin hinge region polypeptide, (ii) an immunoglobulin heavychain CH2 constant region fused to the hinge region, and (iii) animmunoglobulin heavy chain CH3 constant region fused to the CH2 constantregion. The binding domain polypeptide can be a heavy chain variableregion or a light chain variable region.

The binding-domain immunoglobulin fusion proteins are further disclosedin US 2003/0118592 and US 2003/0133939. These antibody fragments areobtained using conventional techniques known to those with skill in theart, and the fragments are screened for utility in the same manner asare intact antibodies.

The term “bispecific molecule” is intended to include any agent, e.g., aprotein, peptide, or protein or peptide complex, which has two differentbinding specificities. For example, the molecule may bind to, orinteract with (a) a cell surface antigen, and (b) an Fc receptor on thesurface of an effector cell. The term “multispecific molecule” or“heterospecific molecule” is intended to include any agent, e.g., aprotein, peptide, or protein or peptide complex, which has more than twodifferent binding specificities. For example, the molecule may bind to,or interact with (a) a cell surface antigen, (b) an Fc receptor on thesurface of an effector cell, and (c) at least one other component.Accordingly, the invention includes, but is not limited to, bispecific,trispecific, tetraspecific, and other multispecific molecules which aredirected to CLDN18.2, and to other targets, such as Fc receptors oneffector cells. The term “bispecific antibodies” also includesdiabodies. Diabodies are bivalent, bispecific antibodies in which the VHand VL domains are expressed on a single polypeptide chain, but using alinker that is too short to allow for pairing between the two domains onthe same chain, thereby forcing the domains to pair with complementarydomains of another chain and creating two antigen binding sites (seee.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2: 1121-1123).

An antibody may be conjugated to a therapeutic moiety or agent, such asa cytotoxin, a drug (e.g., an immunosuppressant) or a radioisotope. Acytotoxin or cytotoxic agent includes any agent that is detrimental toand, in particular, kills cells. Examples include taxol, cytochalasin B,gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof. Suitable therapeutic agents for forming antibodyconjugates include, but are not limited to, antimetabolites (e.g.,methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabin,5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine,thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC), and anti-mitotic agents(e.g., vincristine and vinblastine). In a preferred embodiment, thetherapeutic agent is a cytotoxic agent or a radiotoxic agent. In anotherembodiment, the therapeutic agent is an immunosuppressant. In yetanother embodiment, the therapeutic agent is GM-CSF. In a preferredembodiment, the therapeutic agent is doxorubicin, cisplatin, bleomycin,sulfate, carmustine, chlorambucil, cyclophosphamide or ricin A.

Antibodies also can be conjugated to a radioisotope, e.g., iodine-131,yttrium-90 or indium-111, to generate cytotoxic radiopharmaceuticals.

The antibody conjugates of the invention can be used to modify a givenbiological response, and the drug moiety is not to be construed aslimited to classical chemical therapeutic agents. For example, the drugmoiety may be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, an enzymaticallyactive toxin, or active fragment thereof, such as abrin, ricin A,pseudomonas exotoxin, or diphtheria toxin; a protein such as tumornecrosis factor or interferon-γ; or, biological response modifiers suchas, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol, Rev., 62: 119-58 (1982).

As used herein, an antibody is “derived from” a particular germlinesequence if the antibody is obtained from a system by immunizing ananimal or by screening an immunoglobulin gene library, and wherein theselected antibody is at least 90%, more preferably at least 95%, evenmore preferably at least 96%, 97%, 98%, or 99% identical in amino acidsequence to the amino acid sequence encoded by the germlineimmunoglobulin gene. Typically, an antibody derived from a particulargermline sequence will display no more than 10 amino acid differences,more preferably, no more than 5, or even more preferably, no more than4, 3, 2, or 1 amino acid difference from the amino acid sequence encodedby the germline immunoglobulin gene.

As used herein, the term “heteroantibodies” refers to two or moreantibodies, derivatives thereof, or antigen binding regions linkedtogether, at least two of which have different specificities. Thesedifferent specificities include a binding specificity for an Fc receptoron an effector cell, and a binding specificity for an antigen or epitopeon a target cell, e.g., a tumor cell.

The antibodies described herein may be monoclonal antibodies. The term“monoclonal antibody” as used herein refers to a preparation of antibodymolecules of single molecular composition. A monoclonal antibodydisplays a single binding specificity and affinity. In one embodiment,the monoclonal antibodies are produced by a hybridoma which includes a Bcell obtained from a non-human animal, e.g., mouse, fused to animmortalized cell.

The antibodies described herein may be recombinant antibodies. The term“recombinant antibody”, as used herein, includes all antibodies that areprepared, expressed, created or isolated by recombinant means, such as(a) antibodies isolated from an animal (e.g., a mouse) that istransgenic or transchromosomal with respect to the immunoglobulin genesor a hybridoma prepared therefrom, (b) antibodies isolated from a hostcell transformed to express the antibody, e.g., from a transfectoma, (c)antibodies isolated from a recombinant, combinatorial antibody library,and (d) antibodies prepared, expressed, created or isolated by any othermeans that involve splicing of immunoglobulin gene sequences to otherDNA sequences.

Antibodies described herein may be derived from different species,including but not limited to mouse, rat, rabbit, guinea pig and human.

Antibodies described herein include polyclonal and monoclonal antibodiesand include IgA such as IgA1 or IgA2, IgG1, IgG2, IgG3, IgG4, IgE, IgM,and IgD antibodies. In various embodiments, the antibody is an IgG1antibody, more particularly an IgG1, kappa or IgG1, lambda isotype (i.e.IgG1, κ, λ), an IgG2a antibody (e.g. IgG2a, κ, λ), an IgG2b antibody(e.g. IgG2b, κ, λ), an IgG3 antibody (e.g. IgG3, κ, λ) or an IgG4antibody (e.g. IgG4, κ, λ).

The term “transfectoma”, as used herein, includes recombinant eukaryotichost cells expressing an antibody, such as CHO cells, NS/0 cells, HEK293cells, HEK293T cells, plant cells, or fungi, including yeast cells.

As used herein, a “heterologous antibody” is defined in relation to atransgenic organism producing such an antibody. This term refers to anantibody having an amino acid sequence or an encoding nucleic acidsequence corresponding to that found in an organism not consisting ofthe transgenic organism, and being generally derived from a speciesother than the transgenic organism.

As used herein, a “heterohybrid antibody” refers to an antibody havinglight and heavy chains of different organismal origins. For example, anantibody having a human heavy chain associated with a murine light chainis a heterohybrid antibody.

The invention includes all antibodies and derivatives of antibodies asdescribed herein which for the purposes of the invention are encompassedby the term “antibody”. The term “antibody derivatives” refers to anymodified form of an antibody, e.g., a conjugate of the antibody andanother agent or antibody, or an antibody fragment.

The antibodies described herein are preferably isolated. An “isolatedantibody” as used herein, is intended to refer to an antibody which issubstantially free of other antibodies having different antigenicspecificities (e.g., an isolated antibody that specifically binds toCLDN18.2 is substantially free of antibodies that specifically bindantigens other than CLDN18.2). An isolated antibody that specificallybinds to an epitope, isoform or variant of human CLDN18.2 may, however,have cross-reactivity to other related antigens, e.g., from otherspecies (e.g., CLDN18.2 species homologs). Moreover, an isolatedantibody may be substantially free of other cellular material and/orchemicals. In one embodiment of the invention, a combination of“isolated” monoclonal antibodies relates to antibodies having differentspecificities and being combined in a well defined composition ormixture.

The term “binding” according to the invention preferably relates to aspecific binding.

According to the present invention, an antibody is capable of binding toa predetermined target if it has a significant affinity for saidpredetermined target and binds to said predetermined target in standardassays. “Affinity” or “binding affinity” is often measured byequilibrium dissociation constant (K_(D)). Preferably, the term“significant affinity” refers to the binding to a predetermined targetwith a dissociation constant (K_(D)) of 10⁻⁵ M or lower, 10⁻⁶ M orlower, 10⁻⁷ M or lower, 10⁻⁸M or lower, 10⁻⁹M or lower, 10⁻¹° M orlower, 10⁻¹¹ M or lower, or 10⁻¹²M or lower.

An antibody is not (substantially) capable of binding to a target if ithas no significant affinity for said target and does not bindsignificantly, in particular does not bind detectably, to said target instandard assays. Preferably, the antibody does not detectably bind tosaid target if present in a concentration of up to 2, preferably 10,more preferably 20, in particular 50 or 100 μg/ml or higher. Preferably,an antibody has no significant affinity for a target if it binds to saidtarget with a K_(D) that is at least 10-fold, 100-fold, 10³-fold,10⁴-fold, 10⁵-fold, or 10⁶-fold higher than the K_(D) for binding to thepredetermined target to which the antibody is capable of binding. Forexample, if the K_(D) for binding of an antibody to the target to whichthe antibody is capable of binding is 10⁻⁷ M, the K_(D) for binding to atarget for which the antibody has no significant affinity would be is atleast 10⁻⁶ M, 10⁻⁵ M, 10⁻⁴ M, 10⁻³ M, 10⁻² M, or 10⁻¹ M.

An antibody is specific for a predetermined target if it is capable ofbinding to said predetermined target while it is not capable of bindingto other targets, i.e. has no significant affinity for other targets anddoes not significantly bind to other targets in standard assays.According to the invention, an antibody is specific for CLDN18.2 if itis capable of binding to CLDN18.2 but is not (substantially) capable ofbinding to other targets. Preferably, an antibody is specific forCLDN18.2 if the affinity for and the binding to such other targets doesnot significantly exceed the affinity for or binding toCLDN18.2-unrelated proteins such as bovine serum albumin (BSA), casein,human serum albumin (HSA) or non-claudin transmembrane proteins such asMHC molecules or transferrin receptor or any other specifiedpolypeptide. Preferably, an antibody is specific for a predeterminedtarget if it binds to said target with a K_(D) that is at least 10-fold,100-fold, 10³-fold, 10⁴-fold, 10⁵-fold, or 10⁶-fold lower than the K_(D)for binding to a target for which it is not specific. For example, ifthe K_(D) for binding of an antibody to the target for which it isspecific is 10⁻⁷ M, the K_(D) for binding to a target for which it isnot specific would be at least 10⁻⁶ M, 10⁻⁵ M, 10⁻⁴ M, 10⁻³ M, 10⁻² M,or 10⁻¹ M.

Binding of an antibody to a target can be determined experimentallyusing any suitable method; see, for example, Berzofsky et al.,“Antibody-Antigen Interactions” In Fundamental Immunology, Paul, W. E.,Ed., Raven Press New York, N.Y. (1984), Kuby, Janis Immunology, W. H.Freeman and Company New York, N.Y. (1992), and methods described herein.Affinities may be readily determined using conventional techniques, suchas by equilibrium dialysis; by using the BIAcore 2000 instrument, usinggeneral procedures outlined by the manufacturer; by radioimmunoassayusing radiolabeled target antigen; or by another method known to theskilled artisan. The affinity data may be analyzed, for example, by themethod of Scatchard et al., Ann N.Y. Acad. ScL, 51:660 (1949). Themeasured affinity of a particular antibody-antigen interaction can varyif measured under different conditions, e.g., salt concentration, pH.Thus, measurements of affinity and other antigen-binding parameters,e.g., K_(D), IC₅₀, are preferably made with standardized solutions ofantibody and antigen, and a standardized buffer.

As used herein, “isotype” refers to the antibody. class (e.g., IgM orIgG1) that is encoded by heavy chain constant region genes.

As used herein, “isotype switching” refers to the phenomenon by whichthe class, or isotype, of an antibody changes from one Ig class to oneof the other Ig classes.

The term “naturally occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory isnaturally occurring.

The term “rearranged” as used herein refers to a configuration of aheavy chain or light chain immunoglobulin locus wherein a V segment ispositioned immediately adjacent to a D-J or J segment in a conformationencoding essentially a complete VH or VL domain, respectively. Arearranged immunoglobulin (antibody) gene locus can be identified bycomparison to germline DNA; a rearranged locus will have at least onerecombined heptamer/nonamer homology element.

The term “unrearranged” or “germline configuration” as used herein inreference to a V segment refers to the configuration wherein the Vsegment is not recombined so as to be immediately adjacent to a D or Jsegment.

According to the invention an antibody having the ability of binding toCLDN18.2 is an antibody capable of binding to an epitope present inCLDN18.2, preferably an epitope located within the extracellular domainsof CLDN18.2, in particular the first extracellular domain, preferablyamino acid positions 29 to 78 of CLDN18.2. In particular embodiments, anantibody having the ability of binding to CLDN18.2 is an antibodycapable of binding to (i) an epitope on CLDN18.2 which is not present onCLDN18.1, preferably SEQ ID NO: 3, 4, and 5, (ii) an epitope localizedon the CLDN18.2-loop1, preferably SEQ ID NO: 8, (iii) an epitopelocalized on the CLDN18.2-loop2, preferably SEQ ID NO: 10, (iv) anepitope localized on the CLDN18.2-loopD3, preferably SEQ ID NO: 11, (v)an epitope, which encompass CLDN18.2-loop1 and CLDN18.2-loopD3, or (vi)a non-glycosylated epitope localized on the CLDN18.2-loopD3, preferablySEQ ID NO: 9.

According to the invention an antibody having the ability of binding toCLDN18.2 preferably is an antibody having the ability of binding toCLDN18.2 but not to CLDN18.1. Preferably, an antibody having the abilityof binding to CLDN18.2 is specific for CLDN18.2. Preferably, an antibodyhaving the ability of binding to CLDN18.2 preferably is an antibodyhaving the ability of binding to CLDN18.2 expressed on the cell surface.In particular preferred embodiments, an antibody having the ability ofbinding to CLDN18.2 binds to native epitopes of CLDN18.2 present on thesurface of living cells. Preferably, an antibody having the ability ofbinding to CLDN18.2 binds to one or more peptides selected from thegroup consisting of SEQ ID NOs: 1, 3-11, 44, 46, and 48-50. Preferably,an antibody having the ability of binding to CLDN18.2 is specific forthe afore mentioned proteins, peptides or immunogenic fragments orderivatives thereof. An antibody having the ability of binding toCLDN18.2 may be obtained by a method comprising the step of immunizingan animal with a protein or peptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 1, 3-11, 44, 46, and48-50, or a nucleic acid or host cell expressing said protein orpeptide. Preferably, the antibody binds to cancer cells, in particularcells of the cancer types mentioned above and, preferably, does not bindsubstantially to non-cancerous cells.

Preferably, binding of an antibody having the ability of binding toCLDN18.2 to cells expressing CLDN18.2 induces or mediates killing ofcells expressing CLDN18.2. The cells expressing CLDN18.2 are preferablycancer cells and are, in particular, selected from the group consistingof tumorigenic gastric, esophageal, pancreatic, lung, ovarian, colon,hepatic, head-neck, and gallbladder cancer cells. Preferably, theantibody induces or mediates killing of cells by inducing one or more ofcomplement dependent cytotoxicity (CDC) mediated lysis, antibodydependent cellular cytotoxicity (ADCC) mediated lysis, apoptosis, andinhibition of proliferation of cells expressing CLDN18.2. Preferably,ADCC mediated lysis of cells takes place in the presence of effectorcells, which in particular embodiments are selected from the groupconsisting of monocytes, mononuclear cells, NK cells and PMNs.Inhibiting proliferation of cells can be measured in vitro bydetermining proliferation of cells in an assay using bromodeoxyuridine(5-bromo-2-deoxyuridine, BrdU). BrdU is a synthetic nucleoside which isan analogue of thymidine and can be incorporated into the newlysynthesized DNA of replicating cells (during the S phase of the cellcycle), substituting for thymidine during DNA replication. Detecting theincorporated chemical using, for example, antibodies specific for BrdUindicates cells that were actively replicating their DNA.

In preferred embodiments, antibodies described herein can becharacterized by one or more of the following properties:

-   -   a) specificity for CLDN18.2;    -   b) a binding affinity to CLDN18.2 of about 100 nM or less,        preferably, about 5-10 nM or less and, more preferably, about        1-3 nM or less,    -   c) the ability to induce or mediate CDC on CLDN18.2 positive        cells;    -   d) the ability to induce or mediate ADCC on CLDN18.2 positive        cells;    -   e) the ability to inhibit the growth of CLDN18.2 positive cells;    -   f) the ability to induce apoptosis of CLDN18.2 positive cells.

In a particularly preferred embodiment, an antibody having the abilityof binding to CLDN18.2 is produced by a hybridoma deposited at the DSMZ(Mascheroder Weg 1b, 31824 Braunschweig, Germany; new address:Inhoffenstr. 7B, 31824 Braunschweig, Germany) and having the followingdesignation and accession number:

-   -   a. 182-D1106-055, accession no. DSM ACC2737, deposited on Oct.        19, 2005    -   b. 182-D1106-056, accession no. DSM ACC2738, deposited on Oct.        19, 2005    -   c. 182-D1106-057, accession no. DSM ACC2739, deposited on Oct.        19, 2005    -   d. 182-D1106-058, accession no. DSM ACC2740, deposited on Oct.        19, 2005    -   e. 182-D1106-059, accession no. DSM ACC2741, deposited on Oct.        19, 2005    -   f. 182-D1106-062, accession no. DSM ACC2742, deposited on Oct.        19, 2005,    -   g. 182-D1106-067, accession no. DSM ACC2743, deposited on Oct.        19, 2005    -   h. 182-D758-035, accession no. DSM ACC2745, deposited on Nov.        17, 2005    -   i. 182-D758-036, accession no. DSM ACC2746, deposited on Nov.        17, 2005    -   j. 182-D758-040, accession no. DSM ACC2747, deposited on Nov.        17, 2005    -   k. 182-D1106-061, accession no. DSM ACC2748, deposited on Nov.        17, 2005    -   l. 182-D1106-279, accession no. DSM ACC2808, deposited on Oct.        26, 2006    -   m. 182-D1106-294, accession no. DSM ACC2809, deposited on Oct.        26, 2006,    -   n. 182-D1106-362, accession no. DSM ACC2810, deposited on Oct.        26, 2006.

Preferred antibodies according to the invention are those produced byand obtainable from the above-described hybridomas; i.e. 37G11 in thecase of 182-D1106-055, 37H8 in the case of 182-D1106-056, 38G5 in thecase of 182-D1106-057, 38H3 in the case of 182-D1106-058, 39F11 in thecase of 182-D1106-059, 43A11 in the case of 182-D1106-062, 61C2 in thecase of 182-D1106-067, 26B5 in the case of 182-D758-035, 26D12 in thecase of 182-D758-036, 28D10 in the case of 182-D758-040, 42E12 in thecase of 182-D1106-061, 125E1 in the case of 182-D1106-279, 163E12 in thecase of 182-D1106-294, and 175D10 in the case of 182-D1106-362; and thechimerized and humanized forms thereof.

Preferred chimerized antibodies and their sequences are shown in thefollowing table.

chimerized clone mAb Isotype variable region antibody heavy chain 43A11182-D1106-062 IgG2a SEQ ID NO: 29 SEQ ID NO: 14 163E12 182-D1106-294IgG3 SEQ ID NO: 30 SEQ ID NO: 15 125E1 182-D1106-279 IgG2a SEQ ID NO: 31SEQ ID NO: 16 166E2 182-D1106-308 IgG3 SEQ ID NO: 33 SEQ ID NO: 18175D10 182-D1106-362 IgG1 SEQ ID NO: 32 SEQ ID NO: 17 45C1 182-D758-187IgG2a SEQ ID NO: 34 SEQ ID NO: 19 light chain 43A11 182-D1106-062 IgKSEQ ID NO: 36 SEQ ID NO: 21 163E12 182-D1106-294 IgK SEQ ID NO: 35 SEQID NO: 20 125E1 182-D1106-279 IgK SEQ ID NO: 37 SEQ ID NO: 22 166E2182-D1106-308 IgK SEQ ID NO: 40 SEQ ID NO: 25 175D10 182-D1106-362 IgKSEQ ID NO: 39 SEQ ID NO: 24 45C1 182-D758-187 IgK SEQ ID NO: 38 SEQ IDNO: 23 45C1 182-D758-187 IgK SEQ ID NO: 41 SEQ ID NO: 26 45C1182-D758-187 IgK SEQ ID NO: 42 SEQ ID NO: 27 45C1 182-D758-187 IgK SEQID NO: 43 SEQ ID NO: 28

In preferred embodiments, antibodies, in particular chimerised forms ofantibodies according to the invention include antibodies comprising aheavy chain constant region (CH) comprising an amino acid sequencederived from a human heavy chain constant region such as the amino acidsequence represented by SEQ ID NO: 13 or a fragment thereof. In furtherpreferred embodiments, antibodies, in particular chimerised forms ofantibodies according to the invention include antibodies comprising alight chain constant region (CL) comprising an amino acid sequencederived from a human light chain constant region such as the amino acidsequence represented by SEQ ID NO: 12 or a fragment thereof. In aparticular preferred embodiment, antibodies, in particular chimerisedforms of antibodies according to the invention include antibodies whichcomprise a CH comprising an amino acid sequence derived from a human CHsuch as the amino acid sequence represented by SEQ ID NO: 13 or afragment thereof and which comprise a CL comprising an amino acidsequence derived from a human CL such as the amino acid sequencerepresented by SEQ ID NO: 12 or a fragment thereof.

In one embodiment, an antibody having the ability of binding to CLDN18.2is a chimeric mouse/human IgG1 monoclonal antibody comprising kappa,murine variable light chain, human kappa light chain constant regionallotype Km(3), murine heavy chain variable region, human IgG1 constantregion, allotype Glm(3).

In certain preferred embodiments, chimerised forms of antibodies includeantibodies comprising a heavy chain comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 14, 15, 16, 17, 18,19, and a fragment thereof and/or comprising a light chain comprising anamino acid sequence selected from the group consisting of SEQ ID NOs:20, 21, 22, 23, 24, 25, 26, 27, 28, and a fragment thereof.

In certain preferred embodiments, chimerised forms of antibodies includeantibodies comprising a combination of heavy chains and light chainsselected from the following possibilities (i) to (ix):

-   -   (i) the heavy chain comprises an amino acid sequence represented        by SEQ ID NO: 14 or a fragment thereof and the light chain        comprises an amino acid sequence represented by SEQ ID NO: 21 or        a fragment thereof,    -   (ii) the heavy chain comprises an amino acid sequence        represented by SEQ ID NO: 15 or a fragment thereof and the light        chain comprises an amino acid sequence represented by SEQ ID NO:        20 or a fragment thereof,    -   (iii) the heavy chain comprises an amino acid sequence        represented by SEQ ID NO: 16 or a fragment thereof and the light        chain comprises an amino acid sequence represented by SEQ ID NO:        22 or a fragment thereof,    -   (iv) the heavy chain comprises an amino acid sequence        represented by SEQ ID NO: 18 or a fragment thereof and the light        chain comprises an amino acid sequence represented by SEQ ID NO:        25 or a fragment thereof,    -   (v) the heavy chain comprises an amino acid sequence represented        by SEQ ID NO: 17 or a fragment thereof and the light chain        comprises an amino acid sequence represented by SEQ ID NO: 24 or        a fragment thereof,    -   (vi) the heavy chain comprises an amino acid sequence        represented by SEQ ID NO: 19 or a fragment thereof and the light        chain comprises an amino acid sequence represented by SEQ ID NO:        23 or a fragment thereof,    -   (vii) the heavy chain comprises an amino acid sequence        represented by SEQ ID NO: 19 or a fragment thereof and the light        chain comprises an amino acid sequence represented by SEQ ID NO:        26 or a fragment thereof,    -   (viii) the heavy chain comprises an amino acid sequence        represented by SEQ ID NO: 19 or a fragment thereof and the light        chain comprises an amino acid sequence represented by SEQ ID NO:        27 or a fragment thereof, and    -   (ix) the heavy chain comprises an amino acid sequence        represented by SEQ ID NO: 19 or a fragment thereof and the light        chain comprises an amino acid sequence represented by SEQ ID NO:        28 or a fragment thereof.

“Fragment” or “fragment of an amino acid sequence” as used above relatesto a part of an antibody sequence, i.e. a sequence which represents theantibody sequence shortened at the N- and/or C-terminus, which when itreplaces said antibody sequence in an antibody retains binding of saidantibody to CLDN18.2 and preferably functions of said antibody asdescribed herein, e.g. CDC mediated lysis or ADCC mediated lysis.Preferably, a fragment of an amino acid sequence comprises at least 80%,preferably at least 90%, 95%, 96%, 97%, 98%, or 99% of the amino acidresidues from said amino acid sequence. A fragment of an amino acidsequence selected from the group consisting of SEQ ID NOs: 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, and 28 preferably relates tosaid sequence wherein 17, 18, 19, 20, 21, 22 or 23 amino acids at theN-terminus are removed.

In a preferred embodiment, an antibody having the ability of binding toCLDN18.2 comprises a heavy chain variable region (VH) comprising anamino acid sequence selected from the group consisting of SEQ ID NOs:29, 30, 31, 32, 33, 34, and a fragment thereof.

In a preferred embodiment, an antibody having the ability of binding toCLDN18.2 comprises a light chain variable region (VL) comprising anamino acid sequence selected from the group consisting of SEQ ID NO: 35,36, 37, 38, 39, 40, 41, 42, 43, and a fragment thereof.

In certain preferred embodiments, an antibody having the ability ofbinding to CLDN18.2 comprises a combination of heavy chain variableregion (VH) and light chain variable region (VL) selected from thefollowing possibilities (i) to (ix):

-   -   (i) the VH comprises an amino acid sequence represented by SEQ        ID NO: 29 or a fragment thereof and the VL comprises an amino        acid sequence represented by SEQ ID NO: 36 or a fragment        thereof,    -   (ii) the VH comprises an amino acid sequence represented by SEQ        ID NO: 30 or a fragment thereof and the VL comprises an amino        acid sequence represented by SEQ ID NO: 35 or a fragment        thereof,    -   (iii) the VH comprises an amino acid sequence represented by SEQ        ID NO: 31 or a fragment thereof and the VL comprises an amino        acid sequence represented by SEQ ID NO: 37 or a fragment        thereof,    -   (iv) the VH comprises an amino acid sequence represented by SEQ        ID NO: 33 or a fragment thereof and the VL comprises an amino        acid sequence represented by SEQ ID NO: 40 or a fragment        thereof,    -   (v) the VH comprises an amino acid sequence represented by SEQ        ID NO: 32 or a fragment thereof and the VL comprises an amino        acid sequence represented by SEQ ID NO: 39 or a fragment        thereof,    -   (vi) the VH comprises an amino acid sequence represented by SEQ        ID NO: 34 or a fragment thereof and the VL comprises an amino        acid sequence represented by SEQ ID NO: 38 or a fragment        thereof,    -   (vii) the VH comprises an amino acid sequence represented by SEQ        ID NO: 34 or a fragment thereof and the VL comprises an amino        acid sequence represented by SEQ ID NO: 41 or a fragment        thereof,    -   (viii) the VH comprises an amino acid sequence represented by        SEQ ID NO: 34 or a fragment thereof and the VL comprises an        amino acid sequence represented by SEQ ID NO: 42 or a fragment        thereof,    -   (ix) the VH comprises an amino acid sequence represented by SEQ        ID NO: 34 or a fragment thereof and the VL comprises an amino        acid sequence represented by SEQ ID NO: 43 or a fragment        thereof.

In a preferred embodiment, an antibody having the ability of binding toCLDN18.2 comprises a VH comprising a set of complementarity-determiningregions CDR1, CDR2 and CDR3 selected from the following embodiments (i)to (vi):

-   -   (i) CDR1: positions 45-52 of SEQ ID NO: 14, CDR2: positions        70-77 of SEQ ID NO: 14, CDR3: positions 116-125 of SEQ ID NO:        14,    -   (ii) CDR1: positions 45-52 of SEQ ID NO: 15, CDR2: positions        70-77 of SEQ ID NO: 15, CDR3: positions 116-126 of SEQ ID NO:        15,    -   (iii) CDR1: positions 45-52 of SEQ ID NO: 16, CDR2: positions        70-77 of SEQ ID NO: 16, CDR3: positions 116-124 of SEQ ID NO:        16,    -   (iv) CDR1: positions 45-52 of SEQ ID NO: 17, CDR2: positions        70-77 of SEQ ID NO: 17, CDR3: positions 116-126 of SEQ ID NO:        17,    -   (v) CDR1: positions 44-51 of SEQ ID NO: 18, CDR2: positions        69-76 of SEQ ID NO: 18, CDR3: positions 115-125 of SEQ ID NO:        18, and    -   (vi) CDR1: positions 45-53 of SEQ ID NO: 19, CDR2: positions        71-78 of SEQ ID NO: 19, CDR3: positions 117-128 of SEQ ID NO:        19.

In a preferred embodiment, an antibody having the ability of binding toCLDN18.2 comprises a VL comprising a set of complementarity-determiningregions CDR1, CDR2 and CDR3 selected from the following embodiments (i)to (ix):

-   -   (i) CDR1: positions 47-58 of SEQ ID NO: 20, CDR2: positions        76-78 of SEQ ID NO: 20, CDR3: positions 115-123 of SEQ ID NO:        20,    -   (ii) CDR1: positions 49-53 of SEQ ID NO: 21, CDR2: positions        71-73 of SEQ ID NO: 21, CDR3: positions 110-118 of SEQ ID NO:        21,    -   (iii) CDR1: positions 47-52 of SEQ ID NO: 22, CDR2: positions        70-72 of SEQ ID NO: 22, CDR3: positions 109-117 of SEQ ID NO:        22,    -   (iv) CDR1: positions 47-58 of SEQ ID NO: 23, CDR2: positions        76-78 of SEQ ID NO: 23, CDR3: positions 115-123 of SEQ ID NO:        23,    -   (v) CDR1: positions 47-58 of SEQ ID NO: 24, CDR2: positions        76-78 of SEQ ID NO: 24, CDR3: positions 115-123 of SEQ ID NO:        24,    -   (vi) CDR1: positions 47-58 of SEQ ID NO: 25, CDR2: positions        76-78 of SEQ ID NO: 25, CDR3: positions 115-122 of SEQ ID NO:        25,    -   (vii) CDR1: positions 47-58 of SEQ ID NO: 26, CDR2: positions        76-78 of SEQ ID NO: 26, CDR3: positions 115-123 of SEQ ID NO:        26,    -   (viii) CDR1: positions 47-58 of SEQ ID NO: 27, CDR2: positions        76-78 of SEQ ID NO: 27, CDR3: positions 115-123 of SEQ ID NO:        27, and    -   (ix) CDR1: positions 47-52 of SEQ ID NO: 28, CDR2: positions        70-72 of SEQ ID NO: 28, CDR3: positions 109-117 of SEQ ID NO:        28.

In a preferred embodiment, an antibody having the ability of binding toCLDN18.2 comprises a combination of VH and VL each comprising a set ofcomplementarity-determining regions CDR1, CDR2 and CDR3 selected fromthe following embodiments (i) to (ix):

-   -   (i) VH: CDR1: positions 45-52 of SEQ ID NO: 14, CDR2: positions        70-77 of SEQ ID NO: 14, CDR3: positions 116-125 of SEQ ID NO:        14, VL: CDR1: positions 49-53 of SEQ ID NO: 21, CDR2: positions        71-73 of SEQ ID NO: 21, CDR3: positions 110-118 of SEQ ID NO:        21,    -   (ii) VH: CDR1: positions 45-52 of SEQ ID NO: 15, CDR2: positions        70-77 of SEQ ID NO: 15, CDR3: positions 116-126 of SEQ ID NO:        15, VL: CDR1: positions 47-58 of SEQ ID NO: 20, CDR2: positions        76-78 of SEQ ID NO: 20, CDR3: positions 115-123 of SEQ ID NO:        20,    -   (iii) VH: CDR1: positions 45-52 of SEQ ID NO: 16, CDR2:        positions 70-77 of SEQ ID NO: 16, CDR3: positions 116-124 of SEQ        ID NO: 16, VL: CDR1: positions 47-52 of SEQ ID NO: 22, CDR2:        positions 70-72 of SEQ ID NO: 22, CDR3: positions 109-117 of SEQ        ID NO: 22,    -   (iv) VH: CDR1: positions 44-51 of SEQ ID NO: 18, CDR2: positions        69-76 of SEQ ID NO: 18, CDR3: positions 115-125 of SEQ ID NO:        18, VL: CDR1: positions 47-58 of SEQ ID NO: 25, CDR2: positions        76-78 of SEQ ID NO: 25, CDR3: positions 115-122 of SEQ ID NO:        25,    -   (v) VH: CDR1: positions 45-52 of SEQ ID NO: 17, CDR2: positions        70-77 of SEQ ID NO: 17, CDR3: positions 116-126 of SEQ ID NO:        17, VL: CDR1: positions 47-58 of SEQ ID NO: 24, CDR2: positions        76-78 of SEQ ID NO: 24, CDR3: positions 115-123 of SEQ ID NO:        24,    -   (vi) VH: CDR1: positions 45-53 of SEQ ID NO: 19, CDR2: positions        71-78 of SEQ ID NO: 19, CDR3: positions 117-128 of SEQ ID NO:        19, VL: CDR1: positions 47-58 of SEQ ID NO: 23, CDR2: positions        76-78 of SEQ ID NO: 23, CDR3: positions 115-123 of SEQ ID NO:        23,    -   (vii) VH: CDR1: positions 45-53 of SEQ ID NO: 19, CDR2:        positions 71-78 of SEQ ID NO: 19, CDR3: positions 117-128 of SEQ        ID NO: 19, VL: CDR1: positions 47-58 of SEQ ID NO: 26, CDR2:        positions 76-78 of SEQ ID NO: 26, CDR3: positions 115-123 of SEQ        ID NO: 26,    -   (viii) VH: CDR1: positions 45-53 of SEQ ID NO: 19, CDR2:        positions 71-78 of SEQ ID NO: 19, CDR3: positions 117-128 of SEQ        ID NO: 19, VL: CDR1: positions 47-58 of SEQ ID NO: 27, CDR2:        positions 76-78 of SEQ ID NO: 27, CDR3: positions 115-123 of SEQ        ID NO: 27, and    -   (ix) VH: CDR1: positions 45-53 of SEQ ID NO: 19, CDR2: positions        71-78 of SEQ ID NO: 19, CDR3: positions 117-128 of SEQ ID NO:        19, VL: CDR1: positions 47-52 of SEQ ID NO: 28, CDR2: positions        70-72 of SEQ ID NO: 28, CDR3: positions 109-117 of SEQ ID NO:        28.

In further preferred embodiments, an antibody having the ability ofbinding to CLDN18.2 preferably comprises one or more of thecomplementarity-determining regions (CDRs), preferably at least the CDR3variable region, of the heavy chain variable region (VH) and/or of thelight chain variable region (VL) of a monoclonal antibody againstCLDN18.2, preferably of a monoclonal antibody against CLDN18.2 describedherein, and preferably comprises one or more of thecomplementarity-determining regions (CDRs), preferably at least the CDR3variable region, of the heavy chain variable regions (VH) and/or lightchain variable regions (VL) described herein. In one embodiment said oneor more of the complementarity-determining regions (CDRs) are selectedfrom a set of complementarity-determining regions CDR1, CDR2 and CDR3described herein. In a particularly preferred embodiment, an antibodyhaving the ability of binding to CLDN18.2 preferably comprises thecomplementarity-determining regions CDR1, CDR2 and CDR3 of the heavychain variable region (VH) and/or of the light chain variable region(VL) of a monoclonal antibody against CLDN18.2, preferably of amonoclonal antibody against CLDN18.2 described herein, and preferablycomprises the complementarity-determining regions CDR1, CDR2 and CDR3 ofthe heavy chain variable regions (VH) and/or light chain variableregions (VL) described herein.

In one embodiment an antibody comprising one or more CDRs, a set of CDRsor a combination of sets of CDRs as described herein comprises said CDRstogether with their intervening framework regions. Preferably, theportion will also include at least about 50% of either or both of thefirst and fourth framework regions, the 50% being the C-terminal 50% ofthe first framework region and the N-terminal 50% of the fourthframework region. Construction of antibodies made by recombinant DNAtechniques may result in the introduction of residues N- or C-terminalto the variable regions encoded by linkers introduced to facilitatecloning or other manipulation steps, including the introduction oflinkers to join variable regions of the invention to further proteinsequences including immunoglobulin heavy chains, other variable domains(for example in the production of diabodies) or protein labels.

In one embodiment an antibody comprising one or more CDRs, a set of CDRsor a combination of sets of CDRs as described herein comprises said CDRsin a human antibody framework.

Reference herein to an antibody comprising with respect to the heavychain thereof a particular chain, or a particular region or sequencepreferably relates to the situation wherein all heavy chains of saidantibody comprise said particular chain, region or sequence. Thisapplies correspondingly to the light chain of an antibody.

The term “nucleic acid”, as used herein, is intended to include DNA andRNA. A nucleic acid may be single-stranded or double-stranded, butpreferably is double-stranded DNA.

According to the invention, the term “expression” is used in its mostgeneral meaning and comprises the production of RNA or of RNA andprotein/peptide. It also comprises partial expression of nucleic acids.Furthermore, expression may be carried out transiently or stably.

The teaching given herein with respect to specific amino acid sequences,e.g. those shown in the sequence listing, is to be construed so as toalso relate to variants of said specific sequences resulting insequences which are functionally equivalent to said specific sequences,e.g. amino acid sequences exhibiting properties identical or similar tothose of the specific amino acid sequences. One important property is toretain binding of an antibody to its target or to sustain effectorfunctions of an antibody. Preferably, a sequence which is a variant withrespect to a specific sequence, when it replaces the specific sequencein an antibody retains binding of said antibody to CLDN18.2 andpreferably functions of said antibody as described herein, e.g. CDCmediated lysis or ADCC mediated lysis.

It will be appreciated by those skilled in the art that in particularthe sequences of the CDR, hypervariable and variable regions can bemodified without losing the ability to bind CLDN18.2. For example, CDRregions will be either identical or highly homologous to the regions ofantibodies specified herein. By “highly homologous” it is contemplatedthat from 1 to 5, preferably from 1 to 4, such as 1 to 3 or 1 or 2substitutions may be made in the CDRs. In addition, the hypervariableand variable regions may be modified so that they show substantialhomology with the regions of antibodies specifically disclosed herein.

For the purposes of the present invention, “variants” of an amino acidsequence comprise amino acid insertion variants, amino acid additionvariants, amino acid deletion variants and/or amino acid substitutionvariants. Amino acid deletion variants that comprise the deletion at theN-terminal and/or C-terminal end of the protein are also calledN-terminal and/or C-terminal truncation variants.

Amino acid insertion variants comprise insertions of single or two ormore amino acids in a particular amino acid sequence. In the case ofamino acid sequence variants having an insertion, one or more amino acidresidues are inserted into a particular site in an amino acid sequence,although random insertion with appropriate screening of the resultingproduct is also possible.

Amino acid addition variants comprise amino- and/or carboxy-terminalfusions of one or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50,or more amino acids.

Amino acid deletion variants are characterized by the removal of one ormore amino acids from the sequence, such as by removal of 1, 2, 3, 5,10, 20, 30, 50, or more amino acids. The deletions may be in anyposition of the protein.

Amino acid substitution variants are characterized by at least oneresidue in the sequence being removed and another residue being insertedin its place. Preference is given to the modifications being inpositions in the amino acid sequence which are not conserved betweenhomologous proteins or peptides and/or to replacing amino acids withother ones having similar properties. Preferably, amino acid changes inprotein variants are conservative amino acid changes, i.e.,substitutions of similarly charged or uncharged amino acids. Aconservative amino acid change involves substitution of one of a familyof amino acids which are related in their side chains. Naturallyoccurring amino acids are generally divided into four families: acidic(aspartate, glutamate), basic (lysine, arginine, histidine), non-polar(alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), and uncharged polar (glycine, asparagine,glutamine, cysteine, serine, threonine, tyrosine) amino acids.Phenylalanine, tryptophan, and tyrosine are sometimes classified jointlyas aromatic amino acids.

Preferably the degree of similarity, preferably identity between a givenamino acid sequence and an amino acid sequence which is a variant ofsaid given amino acid sequence will be at least about 60%, 65%, 70%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99%. The degree of similarity or identity isgiven preferably for an amino acid region which is at least about 10%,at least about 20%, at least about 30%, at least about 40%, at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,at least about 90% or about 100% of the entire length of the referenceamino acid sequence. For example, if the reference amino acid sequenceconsists of 200 amino acids, the degree of similarity or identity isgiven preferably for at least about 20, at least about 40, at leastabout 60, at least about 80, at least about 100, at least about 120, atleast about 140, at least about 160, at least about 180, or about 200amino acids, preferably continuous amino acids. In preferredembodiments, the degree of similarity or identity is given for theentire length of the reference amino acid sequence. The alignment fordetermining sequence similarity, preferably sequence identity can bedone with art known tools, preferably using the best sequence alignment,for example, using Align, using standard settings, preferablyEMBOSS::needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.

“Sequence similarity” indicates the percentage of amino acids thateither are identical or that represent conservative amino acidsubstitutions. “Sequence identity” between two amino acid sequencesindicates the percentage of amino acids that are identical between thesequences.

The term “percentage identity” is intended to denote a percentage ofamino acid residues which are identical between the two sequences to becompared, obtained after the best alignment, this percentage beingpurely statistical and the differences between the two sequences beingdistributed randomly and over their entire length. Sequence comparisonsbetween two amino acid sequences are conventionally carried out bycomparing these sequences after having aligned them optimally, saidcomparison being carried out by segment or by “window of comparison” inorder to identify and compare local regions of sequence similarity. Theoptimal alignment of the sequences for comparison may be produced,besides manually, by means of the local homology algorithm of Smith andWaterman, 1981, Ads App. Math. 2, 482, by means of the local homologyalgorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, by meansof the similarity search method of Pearson and Lipman, 1988, Proc. NatlAcad. Sci. USA 85, 2444, or by means of computer programs which usethese algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA inWisconsin Genetics Software Package, Genetics Computer Group, 575Science Drive, Madison, Wis.).

The percentage identity is calculated by determining the number ofidentical positions between the two sequences being compared, dividingthis number by the number of positions compared and multiplying theresult obtained by 100 so as to obtain the percentage identity betweenthese two sequences.

The term “transgenic animal” refers to an animal having a genomecomprising one or more transgenes, preferably heavy and/or light chaintransgenes, or transchromosomes (either integrated or non-integratedinto the animal's natural genomic DNA) and which is preferably capableof expressing the transgenes. For example, a transgenic mouse can have ahuman light chain transgene and either a human heavy chain transgene orhuman heavy chain transchromosome, such that the mouse produces humananti-CLDN18.2 antibodies when immunized with CLDN18.2 antigen and/orcells expressing CLDN18.2. The human heavy chain transgene can beintegrated into the chromosomal DNA of the mouse, as is the case fortransgenic mice, e.g., HuMAb mice, such as HCo7 or HCo12 mice, or thehuman heavy chain transgene can be maintained extrachromosomally, as isthe case for transchromosomal (e.g., KM) mice as described in WO02/43478. Such transgenic and transchromosomal mice may be capable ofproducing multiple isotypes of human monoclonal antibodies to CLDN18.2(e.g., IgG, IgA and/or IgE) by undergoing V-D-J recombination andisotype switching.

“Reduce”, “decrease” or “inhibit” as used herein means an overalldecrease or the ability to cause an overall decrease, preferably of 5%or greater, 10% or greater, 20% or greater, more preferably of 50% orgreater, and most preferably of 75% or greater, in the level, e.g. inthe level of expression or in the level of proliferation of cells.

Terms such as “increase” or “enhance” preferably relate to an increaseor enhancement by about at least 10%, preferably at least 20%,preferably at least 30%, more preferably at least 40%, more preferablyat least 50%, even more preferably at least 80%, and most preferably atleast 100%, at least 200%, at least 500%, at least 1000%, at least10000% or even more.

Mechanisms of mAb Action

Although the following provides considerations regarding the mechanismunderlying the therapeutic efficacy of antibodies of the invention it isnot to be considered as limiting to the invention in any way.

The antibodies described herein preferably interact with components ofthe immune system, preferably through ADCC or CDC. Antibodies describedherein can also be used to target payloads (e.g., radioisotopes, drugsor toxins) to directly kill tumor cells or can be used synergisticallywith traditional chemotherapeutic agents, attacking tumors throughcomplementary mechanisms of action that may include anti-tumor immuneresponses that may have been compromised owing to a chemotherapeutic'scytotoxic side effects on T lymphocytes. However, antibodies describedherein may also exert an effect simply by binding to CLDN18,2 on thecell surface, thus, e.g. blocking proliferation of the cells.

Antibody-Dependent Cell-Mediated Cytotoxicity

ADCC describes the cell-killing ability of effector cells as describedherein, in particular lymphocytes, which preferably requires the targetcell being marked by an antibody.

ADCC preferably occurs when antibodies bind to antigens on tumor cellsand the antibody Fc domains engage Fc receptors (FcR) on the surface ofimmune effector cells. Several families of Fc receptors have beenidentified, and specific cell populations characteristically expressdefined Fc receptors. ADCC can be viewed as a mechanism to directlyinduce a variable degree of immediate tumor destruction that leads toantigen presentation and the induction of tumor-directed T-cellresponses. Preferably, in vivo induction of ADCC will lead totumor-directed T-cell responses and host-derived antibody responses.

Complement-Dependent Cytotoxicity

CDC is another cell-killing method that can be directed by antibodies.IgM is the most effective isotype for complement activation. IgG1 andIgG3 are also both very effective at directing CDC via the classicalcomplement-activation pathway. Preferably, in this cascade, theformation of antigen-antibody complexes results in the uncloaking ofmultiple C1q binding sites in close proximity on the C_(H)2 domains ofparticipating antibody molecules such as IgG molecules (C1q is one ofthree subcomponents of complement C1). Preferably these uncloaked C1qbinding sites convert the previously low-affinity C1q-IgG interaction toone of high avidity, which triggers a cascade of events involving aseries of other complement proteins and leads to the proteolytic releaseof the effector-cell chemotactic/activating agents C3a and C5a.Preferably, the complement cascade ends in the formation of a membraneattack complex, which creates pores in the cell membrane that facilitatefree passage of water and solutes into and out of the cell.

Production and Testing of Antibodies

Antibodies described herein can be produced by a variety of techniques,including conventional monoclonal antibody methodology, e.g., thestandard somatic cell hybridization technique of Kohler and Milstein,Nature 256: 495 (1975). Although somatic cell hybridization proceduresare preferred, in principle, other techniques for producing monoclonalantibodies can be employed, e.g., viral or oncogenic transformation ofB-lymphocytes or phage display techniques using libraries of antibodygenes.

The preferred animal system for preparing hybridomas that secretemonoclonal antibodies is the murine system. Hybridoma production in themouse is a very well established procedure. Immunization protocols andtechniques for isolation of immunized splenocytes for fusion are knownin the art. Fusion partners (e.g., murine myeloma cells) and fusionprocedures are also known.

Other preferred animal systems for preparing hybridomas that secretemonoclonal antibodies are the rat and the rabbit system (e.g. describedin Spieker-Polet et al., Proc. Natl. Acad. Sci. U.S.A. 92:9348 (1995),see also Rossi et al., Am. J. Clin. Pathol. 124: 295 (2005)).

In yet another preferred embodiment, human monoclonal antibodies can begenerated using transgenic or transchromosomal mice carrying parts ofthe human immune system rather than the mouse system. These transgenicand transchromosomic mice include mice known as HuMAb mice and KM mice,respectively, and are collectively referred to herein as “transgenicmice.” The production of human antibodies in such transgenic mice can beperformed as described in detail for CD20 in WO2004 035607

Yet another strategy for, generating monoclonal antibodies is todirectly isolate genes encoding antibodies from lymphocytes producingantibodies of defined specificity e.g. see Babcock et al., 1996; A novelstrategy for generating monoclonal antibodies from single, isolatedlymphocytes producing antibodies of defined specificities. For detailsof recombinant antibody engineering see also Welschof and Kraus,Recombinant antibodies for cancer therapy ISBN-0-89603-918-8 and BennyK. C. Lo Antibody Engineering ISBN 1-58829-092-1.

To generate antibodies, mice can be immunized with carrier-conjugatedpeptides derived from the antigen sequence, i.e. the sequence againstwhich the antibodies are to be directed, an enriched preparation ofrecombinantly expressed antigen or fragments thereof and/or cellsexpressing the antigen, as described. Alternatively, mice can beimmunized with DNA encoding the antigen or fragments thereof. In theevent that immunizations using a purified or enriched preparation of theantigen do not result in antibodies, mice can also be immunized withcells expressing the antigen, e.g., a cell line, to promote immuneresponses.

The immune response can be monitored over the course of the immunizationprotocol with plasma and serum samples being obtained by tail vein orretroorbital bleeds. Mice with sufficient titers of immunoglobulin canbe used for fusions. Mice can be boosted intraperitonealy orintravenously with antigen expressing cells 3 days before sacrifice andremoval of the spleen to increase the rate of specific antibodysecreting hybridomas.

To generate hybridomas producing monoclonal antibodies, splenocytes andlymph node cells from immunized mice can be isolated and fused to anappropriate immortalized cell line, such as a mouse myeloma cell line.The resulting hybridomas can then be screened for the production ofantigen-specific antibodies. Individual wells can then be screened byELISA for antibody secreting hybridomas. By Immunofluorescence and FACSanalysis using antigen expressing cells, antibodies with specificity forthe antigen can be identified. The antibody secreting hybridomas can bereplated, screened again, and if still positive for monoclonalantibodies can be subcloned by limiting dilution. The stable subclonescan then be cultured in vitro to generate antibody in tissue culturemedium for characterization.

Antibodies also can be produced in a host cell transfectoma using, forexample, a combination of recombinant DNA techniques and genetransfection methods as are well known in the art (Morrison, S. (1985)Science 229: 1202).

For example, in one embodiment, the gene(s) of interest, e.g., antibodygenes, can be ligated into an expression vector such as a eukaryoticexpression plasmid such as used by the GS gene expression systemdisclosed in WO 87/04462, WO 89/01036 and EP 338 841 or other expressionsystems well known in the art. The purified plasmid with the clonedantibody genes can be introduced in eukaryotic host cells such as CHOcells, NS/0 cells, HEK293T cells or HEK293 cells or alternatively othereukaryotic cells like plant derived cells, fungal or yeast cells. Themethod used to introduce these genes can be methods described in the artsuch as electroporation, lipofectine, lipofectamine or others. Afterintroduction of these antibody genes in the host cells, cells expressingthe antibody can be identified and selected. These cells represent thetransfectomas which can then be amplified for their expression level andupscaled to produce antibodies. Recombinant antibodies can be isolatedand purified from these culture supernatants and/or cells.

Alternatively, the cloned antibody genes can be expressed in otherexpression systems, including prokaryotic cells, such as microorganisms,e.g. E. coli. Furthermore, the antibodies can be produced in transgenicnon-human animals, such as in milk from sheep and rabbits or in eggsfrom hens, or in transgenic plants; see e.g. Verma, R., et al. (1998) J.Immunol. Meth. 216: 165-181; Pollock, et al. (1999) J. Immunol. Meth.231: 147-157; and Fischer, R., et al. (1999) Biol. Chem. 380: 825-839.

Chimerization

Murine monoclonal antibodies can be used as therapeutic antibodies inhumans when labeled with toxins or radioactive isotopes. Nonlabeledmurine antibodies are highly immunogenic in man when repetitivelyapplied leading to reduction of the therapeutic effect. The mainimmunogenicity is mediated by the heavy chain constant regions. Theimmunogenicity of murine antibodies in man can be reduced or completelyavoided if respective antibodies are chimerized or humanized. Chimericantibodies are antibodies, the different portions of which are derivedfrom different animal species, such as those having a variable regionderived from a murine antibody and a human immunoglobulin constantregion. Chimerisation of antibodies is achieved by joining of thevariable regions of the murine antibody heavy and light chain with theconstant region of human heavy and light chain (e.g. as described byKraus et al., in Methods in Molecular Biology series, Recombinantantibodies for cancer therapy ISBN-0-89603-918-8). In a preferredembodiment chimeric antibodies are generated by joining humankappa-light chain constant region to murine light chain variable region.In an also preferred embodiment chimeric antibodies can be generated byjoining human lambda-light chain constant region to murine light chainvariable region. The preferred heavy chain constant regions forgeneration of chimeric antibodies are IgG1, IgG3 and IgG4. Otherpreferred heavy chain constant regions for generation of chimericantibodies are IgG2, IgA, IgD and IgM.

Humanization

Antibodies interact with target antigens predominantly through aminoacid residues that are located in the six heavy and light chaincomplementarity determining regions (CDRs). For this reason, the aminoacid sequences within CDRs are more diverse between individualantibodies than sequences outside of CDRs. Because CDR sequences areresponsible for most antibody-antigen interactions, it is possible toexpress recombinant antibodies that mimic the properties of specificnaturally occurring antibodies by constructing expression vectors thatinclude CDR sequences from the specific naturally occurring antibodygrafted onto framework sequences from a different antibody withdifferent properties (see, e.g., Riechmann, L. et al. (1998) Nature 332:323-327; Jones, P. et al. (1986) Nature 321: 522-525; and Queen, C. etal. (1989) Proc. Natl. Acad. Sci. U.S.A 86: 10029-10033). Such frameworksequences can be obtained from public DNA databases that includegermline antibody gene sequences. These germline sequences will differfrom mature antibody gene sequences because they will not includecompletely assembled variable genes, which are formed by V (D) J joiningduring B cell maturation. Germline gene sequences will also differ fromthe sequences of a high affinity secondary repertoire antibody atindividual evenly across the variable region.

The ability of antibodies to bind an antigen can be determined usingstandard binding assays (e.g., ELISA, Western Blot, Immunofluorescenceand flow cytometric analysis).

To purify antibodies, selected hybridomas can be grown in two-literspinner-flasks for monoclonal antibody purification. Alternatively,antibodies can be produced in dialysis based bioreactors. Supernatantscan be filtered and, if necessary, concentrated before affinitychromatography with protein G-sepharose or protein A-sepharose. ElutedIgG can be checked by gel electrophoresis and high performance liquidchromatography to ensure purity. The buffer solution can be exchangedinto PBS, and the concentration can be determined by OD280 using 1.43extinction coefficient. The monoclonal antibodies can be aliquoted andstored at −80° C.

To determine if the selected monoclonal antibodies bind to uniqueepitopes, site-directed or multi-site directed mutagenesis can be used.

To determine the isotype of antibodies, isotype ELISAs with variouscommercial kits (e.g. Zymed, Roche Diagnostics) can be performed. Wellsof microtiter plates can be coated with anti-mouse Ig. After blocking,the plates are reacted with monoclonal antibodies or purified isotypecontrols, at ambient temperature for two hours. The wells can then bereacted with either mouse IgG1, IgG2a, IgG2b or IgG3, IgA or mouseIgM-specific peroxidase-conjugated probes. After washing, the plates canbe developed with ABTS substrate (1 mg/ml) and analyzed at OD of405-650. Alternatively, the IsoStrip Mouse Monoclonal Antibody IsotypingKit (Roche, Cat. No. 1493027) may be used as described by themanufacturer.

In order to demonstrate presence of antibodies in sera of immunized miceor binding of monoclonal antibodies to living cells expressing antigen,flow cytometry can be used. Cell lines expressing naturally or aftertransfection antigen and negative controls lacking antigen expression(grown under standard growth conditions) can be mixed with variousconcentrations of monoclonal antibodies in hybridoma supernatants or inPBS containing 1% FBS, and can be incubated at 4° C. for 30 min. Afterwashing, the APC- or Alexa647-labeled anti IgG antibody can bind toantigen-bound monoclonal antibody under the same conditions as theprimary antibody staining. The samples can be analyzed by flow cytometrywith a FACS instrument using light and side scatter properties to gateon single, living cells. In order to distinguish antigen-specificmonoclonal antibodies from non-specific binders in a single measurement,the method of co-transfection can be employed. Cells transientlytransfected with plasmids encoding antigen and a fluorescent marker canbe stained as described above. Transfected cells can be detected in adifferent fluorescence channel than antibody-stained cells. As themajority of transfected cells express both transgenes, antigen-specificmonoclonal antibodies bind preferentially to fluorescence markerexpressing cells, whereas non-specific antibodies bind in a comparableratio to non-transfected cells. An alternative assay using fluorescencemicroscopy may be used in addition to or instead of the flow cytometryassay. Cells can be stained exactly as described above and examined byfluorescence microscopy.

In order to demonstrate presence of antibodies in sera of immunized miceor binding of monoclonal antibodies to living cells expressing antigen,immunofluorescence microscopy analysis can be used. For example, celllines expressing either spontaneously or after transfection antigen andnegative controls lacking antigen expression are grown in chamber slidesunder standard growth conditions in DMEM/F12 medium, supplemented with10% fetal calf serum (FCS), 2 mM L-glutamine, 100 IU/ml penicillin and100 μg/ml streptomycin. Cells can then be fixed with methanol orparaformaldehyde or left untreated. Cells can then be reacted withmonoclonal antibodies against the antigen for 30 min. at 25° C. Afterwashing, cells can be reacted with an Alexa555-labelled anti-mouse IgGsecondary antibody (Molecular Probes) under the same conditions. Cellscan then be examined by fluorescence microscopy.

Cell extracts from cells expressing antigen and appropriate negativecontrols can be prepared and subjected to sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis. After electrophoresis, the separatedantigens will be transferred to nitrocellulose membranes, blocked, andprobed with the monoclonal antibodies to be tested. IgG binding can bedetected using anti-mouse IgG peroxidase and developed with ECLsubstrate.

Antibodies can be further tested for reactivity with antigen byImmunohistochemistry in a manner well known to the skilled person, e.g.using paraformaldehyde or acetone fixed cryosections or paraffinembedded tissue sections fixed with paraformaldehyde from non-cancertissue or cancer tissue samples obtained from patients during routinesurgical procedures or from mice carrying xenografted tumors inoculatedwith cell lines expressing spontaneously or after transfection antigen.For immunostaining, antibodies reactive to antigen can be incubatedfollowed by horseradish-peroxidase conjugated goat anti-mouse or goatanti-rabbit antibodies (DAKO) according to the vendors instructions.

Antibodies can be tested for their ability to mediate phagocytosis andkilling of cells expressing CLDN18.2. The testing of monoclonal antibodyactivity in vitro will provide an initial screening prior to testing invivo models.

Antibody Dependent Cell-Mediated Cytotoxicity (ADCC)

Briefly, polymorphonuclear cells (PMNs), NK cells, monocytes,mononuclear cells or other effector cells, from healthy donors can bepurified by Ficoll Hypaque density centrifugation, followed by lysis ofcontaminating erythrocytes. Washed effector cells can be suspended inRPMI supplemented with 10% heat-inactivated fetal calf serum or,alternatively with 5% heat-inactivated human serum and mixed with ⁵¹Crlabeled target cells expressing CLDN18.2, at various ratios of effectorcells to target cells. Alternatively, the target cells may be labeledwith a fluorescence enhancing ligand (BATDA). A highly fluorescentchelate of Europium with the enhancing ligand which is released fromdead cells can be measured by a fluorometer. Another alternativetechnique may utilize the transfection of target cells with luciferase.Added lucifer yellow may then be oxidated by viable cells only. Purifiedanti-CLDN18.2 IgGs can then be added at various concentrations.Irrelevant human IgG can be used as negative control. Assays can becarried out for 4 to 20 hours at 37° C. depending on the effector celltype used. Samples can be assayed for cytolysis by measuring ⁵¹Crrelease or the presence of the EuTDA chelate in the culture supernatant.Alternatively, luminescence resulting from the oxidation of luciferyellow can be a measure of viable cells.

Anti-CLDN18.2 monoclonal antibodies can also be tested in variouscombinations to determine whether cytolysis is enhanced with multiplemonoclonal antibodies.

Complement Dependent Cytotoxicity (CDC)

Monoclonal anti-CLDN18.2 antibodies can be tested for their ability tomediate CDC using a variety of known techniques. For example, serum forcomplement can be obtained from blood in a manner known to the skilledperson. To determine the CDC activity of mAbs, different methods can beused. ⁵¹Cr release can for example be measured or elevated membranepermeability can be assessed using a propidium iodide (PI) exclusionassay. Briefly, target cells can be washed and 5×10⁵/ml can be incubatedwith various concentrations of mAb for 10-30 min. at room temperature orat 37° C. Serum or plasma can then be added to a final concentration of20% (v/v) and the cells incubated at 37° C. for 20-30 min. All cellsfrom each sample can be added to the PI solution in a FACS tube. Themixture can then be analyzed immediately by flow cytometry analysisusing FACSArray.

In an alternative assay, induction of CDC can be determined on adherentcells. In one embodiment of this assay, cells are seeded 24 h before theassay with a density of 3×10⁴/well in tissue-culture flat-bottommicrotiter plates. The next day growth medium is removed and the cellsare incubated in triplicates with antibodies. Control cells areincubated with growth medium or growth medium containing 0.2% saponinfor the determination of background lysis and maximal lysis,respectively. After incubation for 20 min. at room temperaturesupernatant is removed and 20% (v/v) human plasma or serum in DMEM(prewarmed to 37° C.) is added to the cells and incubated for another 20min. at 37° C. All cells from each sample are added to propidium iodidesolution (10 μg/ml). Then, supernatants are replaced by PBS containing2.5 μg/ml ethidium bromide and fluorescence emission upon excitation at520 nm is measured at 600 nm using a Tecan Safire. The percentagespecific lysis is calculated as follows: % specific lysis=(fluorescencesample-fluorescence background)/(fluorescence maximal lysis-fluorescencebackground)×100.

Induction of Apoptosis and Inhibition of Cell Proliferation byMonoclonal Antibodies

To test for the ability to initiate apoptosis, monoclonal anti-CLDN18.2antibodies can, for example, be incubated with CLDN18.2 positive tumorcells, e.g., SNU-16, DAN-G, KATO-III or CLDN18.2 transfected tumor cellsat 37° C. for about 20 hours. The cells can be harvested, washed inAnnexin-V binding buffer (BD biosciences), and incubated with Annexin Vconjugated with FITC or APC (BD biosciences) for 15 min. in the dark.All cells from each sample can be added to PI solution (10 μg/ml in PBS)in a FACS tube and assessed immediately by flow cytometry (as above).Alternatively, a general inhibition of cell-proliferation by monoclonalantibodies can be detected with commercially available kits. The DELFIACell Proliferation Kit (Perkin-Elmer, Cat. No. AD0200) is a non-isotopicimmunoassay based on the measurement of 5-bromo-2′-deoxyuridine (BrdU)incorporation during DNA synthesis of proliferating cells inmicroplates. Incorporated BrdU is detected using europium labelledmonoclonal antibody. To allow antibody detection, cells are fixed andDNA denatured using Fix solution. Unbound antibody is washed away andDELFIA inducer is added to dissociate europium ions from the labelledantibody into solution, where they form highly fluorescent chelates withcomponents of the DELFIA Inducer. The fluorescence measured—utilizingtime-resolved fluorometry in the detection—is proportional to the DNAsynthesis in the cell of each well.

Preclinical Studies

Monoclonal antibodies which bind to CLDN18.2 also can be tested in an invivo model (e.g. in immune deficient mice carrying xenografted tumorsinoculated with cell lines expressing CLDN18.2, e.g. DAN-G, SNU-16, orKATO-III, or after transfection, e.g. HEK293) to determine theirefficacy in controlling growth of CLDN18.2-expressing tumor cells.

In vivo studies after xenografting CLDN18.2 expressing tumor cells intoimmunocompromised mice or other animals can be performed usingantibodies described herein. Antibodies can be administered to tumorfree mice followed by injection of tumor cells to measure the effects ofthe antibodies to prevent formation of tumors or tumor-related symptoms.Antibodies can be administered to tumor-bearing mice to determine thetherapeutic efficacy of respective antibodies to reduce tumor growth,metastasis or tumor related symptoms. Antibody application can becombined with application of other substances as cystostatic drugs,growth factor inhibitors, cell cycle blockers, angiogenesis inhibitorsor other antibodies to determine synergistic efficacy and potentialtoxicity of combinations. To analyze toxic side effects mediated byantibodies animals can be inoculated with antibodies or control reagentsand thoroughly investigated for symptoms possibly related toCLDN18.2-antibody therapy. Possible side effects of in vivo applicationof CLDN18.2 antibodies particularly include toxicity at CLDN18.2expressing tissues including stomach. Antibodies recognizing CLDN18.2 inhuman and in other species, e.g. mice, are particularly useful topredict potential side effects mediated by application of monoclonalCLDN18.2-antibodies in humans.

Mapping of epitopes recognized by antibodies can be performed asdescribed in detail in “Epitope Mapping Protocols (Methods in MolecularBiology) by Glenn E. Morris ISBN-089603-375-9 and in “Epitope Mapping: APractical Approach” Practical Approach Series, 248 by Olwyn M. R.Westwood, Frank C. Hay.

The compounds and agents described herein may be administered in theform of any suitable pharmaceutical composition.

Pharmaceutical compositions are usually provided in a uniform dosageform and may be prepared in a manner known per se. A pharmaceuticalcomposition may e.g. be in the form of a solution or suspension.

A pharmaceutical composition may comprise salts, buffer substances,preservatives, carriers, diluents and/or excipients all of which arepreferably pharmaceutically acceptable. The term “pharmaceuticallyacceptable” refers to the non-toxicity of a material which does notinteract with the action of the active component of the pharmaceuticalcomposition.

Salts which are not pharmaceutically acceptable may used for preparingpharmaceutically acceptable salts and are included in the invention.Pharmaceutically acceptable salts of this kind comprise in a nonlimiting way those prepared from the following acids: hydrochloric,hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic,citric, formic, malonic, succinic acids, and the like. Pharmaceuticallyacceptable salts may also be prepared as alkali metal salts or alkalineearth metal salts, such as sodium salts, potassium salts or calciumsalts.

Suitable buffer substances for use in a pharmaceutical compositioninclude acetic acid in a salt, citric acid in a salt, boric acid in asalt and phosphoric acid in a salt.

Suitable preservatives for use in a pharmaceutical composition includebenzalkonium chloride, chlorobutanol, paraben and thimerosal.

An injectible formulation may comprise a pharmaceutically acceptableexcipient such as Ringer Lactate.

The term “carrier” refers to an organic or inorganic component, of anatural or synthetic nature, in which the active component is combinedin order to facilitate, enhance or enable application. According to theinvention, the term “carrier” also includes one or more compatible solidor liquid fillers, diluents or encapsulating substances, which aresuitable for administration to a patient.

Possible carrier substances for parenteral administration are e.g.sterile water, Ringer, Ringer lactate, sterile sodium chloride solution,polyalkylene glycols, hydrogenated naphthalenes and, in particular,biocompatible lactide polymers, lactide/glycolide copolymers orpolyoxyethylene/polyoxy-propylene copolymers.

The term “excipient” when used herein is intended to indicate allsubstances which may be present in a pharmaceutical composition andwhich are not active ingredients such as, e.g., carriers, binders,lubricants, thickeners, surface active agents, preservatives,emulsifiers, buffers, flavoring agents, or colorants.

The agents and compositions described herein may be administered via anyconventional route, such as by parenteral administration including byinjection or infusion. Administration is preferably parenterally, e.g.intravenously, intraarterially, subcutaneously, intradermally orintramuscularly.

Compositions suitable for parenteral administration usually comprise asterile aqueous or nonaqueous preparation of the active compound, whichis preferably isotonic to the blood of the recipient. Examples ofcompatible carriers and solvents are Ringer solution and isotonic sodiumchloride solution. In addition, usually sterile, fixed oils are used assolution or suspension medium.

The agents and compositions described herein are administered ineffective amounts. An “effective amount” refers to the amount whichachieves a desired reaction or a desired effect alone or together withfurther doses. In the case of treatment of a particular disease or of aparticular condition, the desired reaction preferably relates toinhibition of the course of the disease. This comprises slowing down theprogress of the disease and, in particular, interrupting or reversingthe progress of the disease. The desired reaction in a treatment of adisease or of a condition may also be delay of the onset or a preventionof the onset of said disease or said condition.

An effective amount of an agent or composition described herein willdepend on the condition to be treated, the severeness of the disease,the individual parameters of the patient, including age, physiologicalcondition, size and weight, the duration of treatment, the type of anaccompanying therapy (if present), the specific route of administrationand similar factors. Accordingly, the doses administered of the agentsdescribed herein may depend on various of such parameters. In the casethat a reaction in a patient is insufficient with an initial dose,higher doses (or effectively higher doses achieved by a different, morelocalized route of administration) may be used.

The agents and compositions described herein can be administered topatients, e.g., in vivo, to treat or prevent a variety of disorders suchas those described herein. Preferred patients include human patientshaving disorders that can be corrected or ameliorated by administeringthe agents and compositions described herein. This includes disordersinvolving cells characterized by an altered expression pattern ofCLDN18.2.

For example, in one embodiment, antibodies described herein can be usedto treat a patient with a cancer disease, e.g., a cancer disease such asdescribed herein characterized by the presence of cancer cellsexpressing CLDN18.2.

The pharmaceutical compositions and methods of treatment describedaccording to the invention may also be used for immunization orvaccination to prevent a disease described herein.

The present invention is further illustrated by the following exampleswhich are not be construed as limiting the scope of the invention.

EXAMPLES Example 1 Material and Methods

1. Antibodies

TABLE 1 Antibodies used herein Provider Binding Antibody (Cat. #) Targetsite Clonality Species Applications IMAB362 Ganymed CLDN18.2 N-Termmonoclonal chimeric ADCC, CDC, IF anti Claudin18 Zymed CLDN18.1 C-termpolyclonal rabbit IF, IHC (MID) #38-8100 CLDN18.2 aa220-246 antiClaudin18 Zymed CLDN18.1 C-term polyclonal rabbit WB (C-term) #38-800CLDN18.2 aa240-262 43-14A Ganymed CLDN18.2 C-term monoclonal mouse IHCaa248-262 35-22A Ganymed CLDN18.2 C-term monoclonal mouse IF, IHCaa248-262 anti human Antibody online human — monoclonal rabbit IHCEPR1394Y MHC-I β-Actin SIGMA Actin monoclonal mouse WB

2. Immunhistochemistry (IHC)

Tissue sections (4 μM thickness) were stored at 2-8° C. until use.

Prior to the deparaffinization process the sections were incubated at58-60° C. in a drying oven for 1 hour to melt the paraffin and toquantitatively remove water, thereby improving adherence of the tissuesto the glass slides (“baking”).

Deparaffinization

After melting and drying, the slides were deparaffinized using two xylolsteps (5 minutes) and rehydrated using a descending alcohol array (at anambient temperature of 20-27° C.):

-   -   5 (±1) minutes in a xylene bath;    -   this step was repeated once in a fresh bath;    -   excess liquid;    -   5 (±1) minutes in absolute ethanol;    -   repeat this step once with a fresh bath;    -   remove excess liquid;    -   5 (±1) minutes in 96% ethanol;    -   repeat this step once with a fresh bath;    -   remove excess liquid;    -   5 (±1) minutes in 80% ethanol;    -   remove excess liquid;    -   5 (±1) minutes in 70% ethanol;    -   remove excess liquid;    -   5 minutes in distilled or deionized water;

Epitope Retrieval and Quenching

Following the paraffin removal the target epitopes were retrieved byusing a heat induced epitope retrieval procedure. Therefore the slideswere put in staining jars filled with 200 ml of retrieval buffer (10 mMCitric buffer; 0.05% Tween-20; pH 6) and incubated in a pressure cooker(PASCAL, Dako) at 120° C. for 10 minutes. Afterwards the jars wereremoved from the cooker and allowed to cool in the Epitope RetrievalSolution for 10 (±1) min at room temperature. The slides were rinsed inWash Buffer (1×PBS).

After cooling the sections were moved into staining jars filled with 200ml quenching solution (0.3% Peroxidase in 1×PBS) and incubated for 15min at room temperature, followed by 2×5 minutes washing steps in freshwash buffer.

Blocking and Antibody Incubation

The excess wash buffer was removed, the slides were covered with 200 μlblocking buffer (10% goat serum in 1×PBS) and incubated at RT for 30minutes. The blocking buffer was removed and replaced by 200 μl dilutedantibody solution (dilution in blocking buffer). The slides wereincubated with the primary antibody overnight at 2-8° C.

TABLE 2Dilution of primary antibodies for histology analysis-stock concentrationand final concentration of the antibodies used in histological assaysPrimary antibody dilution Stock epitope Final Antibody conc. subtypeepitope sequence conc. mumAb 43-14A 1 mg/ml IgG2b CLDN18 c-term.TEDEVQSYPSKHDYV 0.5 μg/mL mumAb 35-22A 1 mg/ml IgG2b CLDN18 c-term.TEDEVQSYPSKHDYV 0.2 μg/mL

On the next day the primary antibody solution was removed and thesections were washed for 3×5 min with washing buffer. Afterwards theexcess wash buffer was removed and 200 μl of the ready-to-use secondaryantibody solution was added (Power Vision HRP goat-α-mouse; Immunologic;NL). The slides were incubated for 30 min at RT. The excess liquid wasremoved and the slides washed for 3×5 min in fresh washing buffer.

Substrate Reaction and Counterstaining

After removal of excess washing buffer the sections were covered withapprox. 50-150 μL of the freshly prepared substrate-chromogen solution(VectorRed; Vector Labs) for 2 min. The excess substrate was removed andthe slides were incubated in jars with deionized water for 1-5 min.Subsequently, a counterstain of the tissue was performed by immersingthe sections in jars containing 200 ml of Mayer's haematoxylin for 2min. Afterwards the sections were placed in tap water for 5-10 min forthe blueing of the nuclei.

Dehydration and Mounting

After performing the counterstaining, the sections were dehydrated usingan ascending alcohol array:

-   -   dipping in 70% ethanol (approx. 5-10 sec)    -   dipping in 80% ethanol (approx. 5-10 sec)    -   dipping in 96% ethanol (approx. 5-10 sec)    -   dipping in 96% ethanol (approx. 5-10 sec)    -   dipping in absolute ethanol (approx. 5-10 sec)    -   5 min in xylene    -   5 min in xylene

For the mounting of the samples a non-aqueous mounting medium(X-TRA-Kit, Medite) was used. The slides were mounted directly from thelast xylene filled jar and air dried at RT.

TABLE 3 Tissue micro arrays for histology analysis number number TMA IDdistributer of cases of cores core size thickness PA961; Biocat 96 961.5 mm 5 μm Pancreatic carcinoma and normal tissue microarray PA802;Biocat 78 80 1.5 mm 5 μm Multiple pancreatic carcinoma tissue array,single core per case BIC14011; Biocat 24 48 1.5 mm 5 μm Pancreasintraepithelial neoplasia tissue microarray

3. Cell Culture

All pancreatic cancer cell lines and additional control cell lines usedfor experiments presented herein are cultivated in media according todata sheets of origin and following standard tissue culture procedures.Conditions are summarized in Table 4. For all newly obtained cell lines,cells were tested for mycoplasm contamination and a master cell bank wasprepared.

TABLE 4 Cell culture conditions for human pancreatic cancer and controlcell lines Seeding density Cell line¹ Flask/dish Medium Incubation 2days² 3 days² AsPC-1 T150 RPMI + 10% FCS 5% CO₂, 37° C. 1e7 8e6 BxPC3T150 RPMI + 10 mM HEPES + 1 mM sodium 5% CO₂, 37° C. 8e6 7e6 (ATCC)pyruvate + 4.5 g/L glucose (total) + 10% FCS Gold BxPC3-LVT T150 RPMI +10 mM HEPES + 1 mM sodium 5% CO₂, 37° C. 1e7 8e6 (ECACC) pyruvate + 1xsodium bicarbonate + 4.5 g/L glucose + 10% FCS + 1% Pen/Strep + 0.5μg/ml (fresh) Blasticidin BxPC3-LVT- T150 RPMI + 10 mM HEPES + 1 mMsodium 5% CO₂, 37° C. 1e7 8e6 Luciferase pyruvate + 1x sodiumbicarbonate + 4.5 g/L glucose + 10% FCS + 1% Pen/Strep + 0.5 μg/mlBlasticidin (fresh) + 40 μg/ml Hygromycin (fresh) CAPAN1 T150 RPMI + 20%FCS 5% CO₂, 37° C. 8e6 8e6 CAPAN1-LVT T150 RPMI + 20% FCS + 1%Pen/Strep + 2.5 μg/ml 5% CO₂, 37° C. 1e7 8e6 Blasticidin (fresh)CAPAN1-LVT- T150 RPMI + 20% FCS + 1% Pen/Strep + 2.5 μg/ml 5% CO₂, 37°C. 1e7 8e6 Blasticidin (fresh) + 40 μg/ml Hygromycin CHO-K1p740 15 cmDMEM:F12 + 1% Pen/Strep + 10% FCS + 7.5% CO₂, 1.6e6 7e5 MACS/FACS 1.5mg/mL Geneticin (G-418) 37° C. (24H5) Luci #2A5 CFPAC-1 T150 Iscove'sMDM + 10% FCS 5% CO₂, 37° C. 5e6 3e6 DANG 1C5F2 15 cm RPMI + 1%Pen/Strep + 10% FCS 5% CO₂, 37° C. 4e6 2e6 DANG 1C5F2 LVT 15 cm RPMI +1% Pen/Strep + 10% FCS + 1 μg/ml 5% CO₂, 37° C. 4e6 2e6 Blasticidin(fresh) HEK293p740#A5 15 cm DMEM:F12 + 1% Pen/Strep + 10% FCS + 7.5%CO₂, 8e6 5e6 1.5 mg/mL Geneticin (G-418) 37° C. HPAC T150 DMEM:F12 + 15mM HEPES + 0.002 mg/ml 5% CO₂, 37° C. 6e6 4e6 human Insulin + 10 ng/mLEGF + 5% FCS HPAC-LVT T150 DMEM:F12 + 15 mM HEPES + 0.002 mg/ml 5% CO₂,37° C. 6e6 4e6 human Insulin + 10 ng/mL EGF + 5% FCS + 1% Pen/Strep +3.5 μg/ml Blasiticidin (fresh) HPAF-II T150 MEM + 10% FCS 5% CO₂, 37° C.5e6 5e6 HUP-T3 T150 MEM + 1x MEM NEAA + 1 mM sodium 5% CO₂, 37° C. 6e63e6 pyruvate + 10% FCS HUP-T4 T150 MEM + 1x MEM NEAA + 1 mM sodium 5%CO₂, 37° C. 6e6 4e6 pyruvate + 20% FCS KATO III FGF-BP T150 RPMI + 1%Pen/Strep + 4 mM Glutamax 7.5% CO₂, 8e6 5e6 #12 adM³ (total) + 20% FCS37° C. KP-2 T150 RPMI + 10% FCS 5% CO₂, 37° C. 8e6 6e6 MiaPaCa-2 T150MEM + 10% FCS 5% CO₂, 37° C. 1e7 8e6 MiaPaCa-2-LVT T150 MEM + 10% FCS +1% Pen/Strep + 1.5 μg/ml 5% CO₂, 37° C. 1e7 8e6 Blasticidin NUGC-4 subT150 RPMI + 1% Pen/Strep + 10% FCS 5% CO₂, 37° C. 8e6 C 5e6 C 10cH11subE10 Luci#2 Panc-1 T150 DMEM + 10% FCS 5% CO₂, 37° C. 6e6 C 4e6 CPanc03.27 T150 RPMI + 10 mM HEPES + 1 mM sodium 5% CO₂, 37° C. 6e6 C 4e6C pyruvate + 4.5 g/L glucose + 0.01 mg/mL Insulin + 15% FCS Panc05.04T150 RPMI + 10 mM HEPES + 1 mM sodium 5% CO₂, 37° C. 6e6 C 4e6 Cpyruvate + 4.5 g/L glucose + 0.01 mg/mL Insulin + 15% FCS Panc05.04 T150RPMI + 10 mM HEPES + 1 mM sodium 5% CO₂, 37° C. 7e6 C 5e6 C subclonespyruvate + 4.5 g/L glucose + 0.01 mg/ml Insulin + 15% FCS Patu8902 T150DMEM + 10% FCS 5% CO₂, 37° C. 5e6 C 4e6 C Patu8902-LVT T150 DMEM + 10%FCS + 1% Pen/Strep + 5% CO₂, 37° C. 5e6 C 4e6 C 9 μg/ml Blasticidin(fresh) Patu8988T T150 DMEM + 5% horse serum + 5% FCS 5% CO₂, 37° C. 3e6C 1e6 C Patu8988S T150 DMEM + 5% horse serum + 5% FCS 5-7.5% CO₂, 1.5e71e7 37° C. Su86.86 T150 RPMI + 10 mM HEPES + 1 mM sodium 5% CO₂, 37° C.5e6 3e6 pyruvate + 4.5 g/L glucose + 10% FCS Suit-2 T150 RPMI + 10% FCS(use new flask for each split) 5% CO₂, 37° C. 1.5e7 1.2e7 Suit-2-LVTT150 RPMI + 10% FCS + 1% Pen/Strep + 5 μg/ml 5% CO₂, 37° C. 1.5e7 1.2e7Blasticidin (fresh) (use new flask for each split) SW1990 T150Leibovitz's L-15 + 10% FCS 37° C. 4e6 3e6 YAPC T150 RPMI + 10% FCS Gold5% CO₂, 37° C. 1.5e7 1e7 YAPC-LVT T150 RPMI + 10% FCS Gold + 1%Pen/Strep + 0.5 μg/ml 5% CO₂, 37° C. 1.5e7 1e7 Blasticidin (fresh) ¹LVTrefers to cell lines stably transduced with lentivirus for expression ofCLDN18.2. ²Seeding density in the mentioned flask or dish forcultivation of cells for 2 or 3 days. ³adM: Cells are-recultivated fromsubcutaneous tumors

4. Luciferase Transfection of Pancreas Cell Lines

For ADCC assays pancreatic cancer cell lines were transientlytransfected with luciferase RNA. This luciferase RNA(pST1-luc2mut-2hBgUTR-A121-EciI vector (pST1-109)) was produced with anARCA cap and dissolved in H₂O. RNA was stored in 22 μl aliquots at −80°C. For all pancreas cell lines the optimal electroporation conditionswere determined resulting in highest transfection rates and viability ofthe cells. In each assay cells were detached with PBS/5 mM EDTA and2.5×10⁶ cells dissolved in 250 μl X-Vivo were mixed in ice-cold cuvetteswith 10 μg RNA. Cells were immediately electroporated (GenePulser Xcell,Biorad) and resuspended in pre-warmed Assay-medium adjusting the cellsto 5×10⁵ cells/ml. Tested electroporation conditions were for all celllines:

-   -   EP1: 250 V, 475 μF    -   EP2: 200 V, 300 μF    -   EP3: 150 V, 300 μF    -   EP4: 200 V, 400 μF    -   EP5: 250 V, 950 μF    -   Control: 0 V, 0 μF

The viability of the cells was determined directly after electroporationusing CASY or by staining cells with Trypan blue and determining thepercentage of dead cells in the Neubauer chamber. Cells were seeded inquadruplicates in white 96-well plates (2.5×10⁴ cells/well) andincubated for 24 h. Subsequently, luciferase activity was measured in aluminometer (Tecan Infinite200) after addition of luciferin mix for 90min. Transfection was successful and consequently ADCC measurable, ifRLU values>1.000 were obtained.

5. Quantitative Real-Time PCR (Q-PCR)

For RNA isolation from pancreatic cancer cell lines, cells were seededin 10 cm dishes and grown for 2-3 days until 80% confluent. RNA wasisolated according to instructions supplied with the RNeasy® Mini Kit(Qiagen). cDNA preparation was performed following manufacturersinstructions provided with the SuperScript® III First Strand kit(Invitrogen). RNA and cDNA samples were stored at −80° C.

Quantitative analysis of CLDN18.2 transcripts was performed byamplifying oligo(dT)-primed cDNAs in a 40 cycle PCR reaction using PCRprimers #5054s (5′-AGAGAGCTCTGGCTTCACCGAGTG-3′) and #5060as(5′-CCAGAAGTTAGTCACCAGCATGTTGG-3′) differentiating between CLDN18.1 andCLDN18.2 isoforms. The reaction was prepared with SYBR Green (QuantiTectSYBR Green PCR Kit, Qiagen), which intercalates in double stranded DNA.The reactions and measurements were performed using the ABI-PRISM7900Sequence Detection System instrument and software (Applied Biosystems).

The relative expression levels of CLDN18 transcripts was computed usingΔΔCT calculation with respect to the house keeping gene HPRT.

6. Western Blot Analysis

For isolation of proteins of pancreatic cancer cell lines, cells wereseeded in 10 cm dishes and grown for 2-3 days until 80% confluent. Cellswere lysed by addition of 800 μl 4×SDS sample buffer (34% glycin, 250 mMTris pH6.8, 5% β-mercaptoethanol, 8.2% SDS). To disintegrate the genomicDNA, the protein samples were sonified under following conditions:Output Control: levell, Duty Cycle: 70% for 20-25 sec. Proteinconcentration was measured, in a spectrophotometer (Absorption at 280nm) and samples were stored at −80° C. until use.

To detect CLDN18.2 expression in Western blots, a 12.5% poly acrylamidegel for separation (for 2 small gels 4.1 ml 29:1acrylamide/bis-acrylamide, 100 μl 10% SDS, 2.5 ml Tris pH8.8, 3.2 mlH₂O, 100 μl APS, 10 μl TEMED) was prepared between two fixed glassplates. After polymerization the gel was overlaid with a stacking gel(1.5 ml 29:1 acrylamide/bis-acrylamide, 100 μl 10% SDS, 2.5 ml TrispH6.8, 5.8 ml H₂O, 100 μl APS, 10 μl TEMED) and a gel comb was placedbetween the glass plates. After polymerization the gel was loaded with75 μg of each protein sample prepared by addition of (1:20) 4×SDS-samplebuffer (250 mM Tris-HCL, 34% Glycerine, 8.2% SDS, pH 6.8) and 7.5 μl ofa size marker-mix (1.5 μl Magic Mark XP Western Standard mixed with 6 μlSeaBlue Plus2 Prestained Standard). Gels were run in 1×SDS runningbuffer (25 mM Tris, 0.192 M Glycin, 0.1% SDS) at 80 V for 30 min and 180V for 60 min. Semi-dry blotting of the gel on a nitrocellulose membranewas performed for 90 min at 160 mA in 1×transfer buffer (25 mM Tris,0.192 mM glycin, 20% MeOH). Blots were first blocked in 5% milkpowder/PBS and primary antibodies (0.25 μg/ml anti-Claudin18 (C-term) or0.1 μg/ml anti-β-actin) were added in a solution of 1% milk powder/PBS.Blots were incubated over night at 4° C., washed 3 times 10 min in1×PBS/0.05% Tween20 and then incubated for 1 h with labelled secondaryantibodies at room temperature in 1% milk powder/PBS (goat-anti-rabbitIgG (FC) diluted 1:1000). Blots were washed again 3 times 10 min in1×PBS/0.05% Tween20 and detection was performed by addition of 1-3 mldetection solution (Pico and Dura Detection System (Pierce) for 1 minand scanning of the blots in a LAS-3000 detection box (Increment: 10sec, Interval Time: 10 sec, Sensitivity: high) according to GA 056Chemolumineszenzentwickler LAS3000.

7. Flowcytometry (FACS)

Cells were harvested with PBS/5 mM EDTA or Trypsin/EDTA from anexponentially growing culture at 70-85% confluency. Cells were counted,centrifuged for 5 min (468 g) and the pellet was resuspended in FACSbuffer (2% FCS, 0.1% sodium azide in PBS) adjusting the concentration to2×10⁶/ml. 100 μl cells were plated in round bottom 96-well plates andagain centrifuged (5 min, 468 g). IMAB362 (or isotype control Rituximab)was serially diluted 0.1-200 μg/ml (11 dilution steps+no antibodycontrol) in 50 μl FACS-buffer and added to the cells for 30 min at 4° C.Then, 200 μl FACS buffer was added to each well and plates werecentrifuged (5 min, 468 g). The supernatant was removed and washing wasrepeated. Secondary goat anti-human antibodies (FC specific, F(ab′)2conjugated with APC (Dianova)) were diluted (1:100) in FACS buffer and30 μl was added to each well. Plates were incubated for 30 min at 4° C.After incubation plates were washed again two times with 200 μl FACSbuffer and the pellet was finally resuspended in 100 μl FACS buffer formeasurement FACS Array Bioanalyzer (BD) according to GA_(—)018_BD FACSArray bioanalyzer.

8. Lentiviral Transduction

Lentiviral Vector Construction:

Lentiviruses belong to the RNA viruses, which stably integrate intohuman genomic DNA of both dividing and non-dividing cells. VectorpLenti6.4 (Invitrogen) was used as backbone. It contains a Blasticidingene for selection of positively transduced cells. CLDN18.2 fused to theEF1α promoter was cloned into the recombination region of the vectorgenerating pL64B42E (EF1α-hCLaudin18.2)-Blasticidin (FIG. 1).

Selection of Cell Lines:

Cell lines were selected according to literature data or, which werepreviously tested in vivo. Selection criteria included homogenoussubcutaneous growth in nude mice and a therapeutic window of 20-100days. Three cell lines (DANG, YAPC and BxPC3) were integrated thatalready showed weak expression of CLDN18.2 mRNA and three (MiaPaCa-2,Patu8902 and Suit-2) that are able to metastasize according toliterature. Two other cell lines (known to grow as homogenoussubcutaneous tumors in vivo) were selected randomly (HPAC, CAPAN1).

Determination of Blasticidin Selection Conditions:

For all cell lines the blasticidin concentration required for selectionof cells after lentiviral transduction was determined beforetransduction was performed. Pancreatic cancer cells were seeded in 6well plates at a high density, resulting in 80-90% confluence after 24h. Blasticidin (Stock: 10 mg/ml, Invitrogen) was added to the wells inincreasing concentrations ranging from 0.5-12 μg/ml (5 dilution steps+noblasticidin control). The medium was exchanged every 3-4 days and cellswere analyzed in a microscope before removing the medium. The amount ofdead cells and the condition of the living cells was documented. Cellswere cultivated for 14 days. The lowest blasticidin concentrationcausing 100% apoptotic cells after 14 days was preferred for selectionof lentivirally transduced cells. The required blasticidinconcentrations for each of the established LVT cell lines are indicatedin Table 4.

Envelope Selection:

For lentiviral transduction, GFP-lentiviral control vectorpL64B42E-(EF1a-GFP)-blast was packaged into different envelope particles(VSV-G, GALV, RD114, Mokola-G and Rabies-G). Depending on the proteinspresent in the envelope and the composition of the cellular membrane,attachment to the target cancer cells is more or less efficient. For allpancreatic cancer cell lines the VSV-G envelope showed highesttransduction efficiency (68.5-91.2%) (Table 5). Consequently, theCLDN18.2 expression vector pL64B42E (EF1α-hCLaudin18.2)-Blasticidin waspackaged into VSV-G envelopes. Producer cells were infected and theviruses were isolated from the medium at high titers (3.86×10⁷particles/ml). Viral supernatants were stored at −80° C.

TABLE 5 Generation of CLDN18.2 over-expressing pancreatic cancer celllines by lentiviral transduction Transduction efficiency Envelope test(GFP control viral vector) of pL64B42E Cell line VSV-G GALV RD114aMokola-G Rabies-G (EF1α-hCLaudin18.2)¹ BxPC3-LVT 91.1 29.5 21.4 61.457.1 92.8% CAPAN1-LVT 83.7 12.3 31.7 24.3 23.1 89.6% DANG-LVT 91.2 41.313.8 33,7 41.4 87.5% HPAC-LVT 77.7 38.4 49.6 72.8 61.5 97.3%MiaPaCa-2-LVT n.d. n.d. n.d. n.d. n.d. 96.3% Patu8902-LVT n.d. n.d. n.d.n.d. n.d. 93.0% Suit2-LVT n.d. n.d. n.d. n.d. n.d. 92.2% YAPC-LVT 68.541.3 13.8 33.7 41.4 82.2% ¹Efficiencies obtained by packaging of thevector in VSV-G envelope particles, measured 2 days after infection.

Lentiviral Transduction of Pancreatic Cancer Cell Lines:

For infection of the pancreatic cancer target cell lines, a 24-wellplate was coated with 200 μl 1× Retronectin® (20 μg/ml, Takara Inc.) andthe plate was sealed with Parafilm® and incubated for 3-16 h at 4° C.Plates were washed with 200 ml PBS and blocked with PBS/2% BSA for 30min at RT. Plates were washed again and loaded with 300 μl viralsupernatant by centrifugation for 25 min at 2500 rpm at 15° C. Thesupernatant was removed and loading was repeated 3 times. The plateswere finally washed once with PBS and target cells at low passage wereseeded in each well. For all pancreatic cancer cell lines, 5×10⁵-1×10⁷cells per 24 well were seeded. Plates were incubated for 2 days at 37°C. Subsequently cells were detached and transduction efficiency wasdetermined by FACS using a FITC-labeled IMAB362 antibody. Cells wereexpanded and a master cell bank was prepared for each cell line.

9. ADCC Assay

Pancreatic cancer target cells were seeded two days in advance in flasksto obtain 80-90% confluent cultures on the day ADCC started. Pancreaticcancer cells were transfected with luciferase RNA and were seeded inwhite 96 well plates at a density of 1×10⁴ cells per well in 50 μl assaymedium (culture medium as described in Table 4 with 20 mM HEPES). NUGC-4sub 10cH11 subE10 Luci#2 cells (8000 cells/well) were seeded in additionas positive controls in all assays. Cells were cultivated for 4-6 hbefore addition of antibody and purified PBMCs.

PBMCs were prepared from fresh human buffy coat obtained from healthydonors. About 3×20-25 ml blood was diluted (1:2) with PBS and carefullylayered on 4×15 ml Ficol-Paque Plus (GE Healthcare) in 50 ml Falcontubes. Gradients were centrifuged (25 min, 700 g). After centrifugation,peripheral blood mononuclear cells (PBMC) were collected from theinterphase, washed in PBS/2 mM EDTA, centrifuged (5 min, 468 g), againresuspended in PBS/2 mM EDTA and centrifuged (10 min, 208 g) to removethe platelets. The pellets were resuspended in 50 ml PBS/2 mM EDTA andcells were counted. PBMCs were centrifuged (5 min, 468 g) andresuspended in X-Vivo-15 culture medium at a concentration of 1.6×10⁷cells/ml for addition to the pancreas cells and 1.28×10⁷ cells/ml foraddition to the NUGC-4 sub 10cH11 subE10 Luci#2 cells.

Antibodies (IMAB362 and the isotype control antibody ch78H11 1H6) wereserially diluted (4.5 fold) 10 times resulting in a concentration rangeof 200 μg/ml-0.26 ng/ml. Of each dilution 25 μl was added inquadruplicates to the target cells. PBS without antibodies was added inthe medium and lysis control wells. Subsequently, 25 μl PBMCs were addedto each well (E:T ratio=40:1) and plates were incubated for 24 h 1 h at37° C., 5% CO₂.

The next day, 10 μl 8% Triton X100/PBS solution was added to the lysiscontrol wells and 10 μl PBS in all other wells. Finally, 50 μl freshlyprepared luciferin stock solution was added (160 mM HEPES, lx PBS, 3.84mg/ml D-Luciferin (BD Biosciences)) to each well and plates wereincubated for 80 min at RT in the dark. Luminescence resulting from theoxidation of Lucifer yellow by the luciferase of viable cells wasmeasured using a microplate-reader (Infinite200, Tecan, Switzerland).Percentage of cellular cytotoxicity was calculated using the followingformula:

Specifickilling(%)=100−[(RLU_(sample)−RLU_(triton))(RLU_(medium ctrl)−RLU_(triton))×100]

10. CDC

CDC was performed as follows.

Target cells (CHO-K1 p740 MACS/FACS (24H5) p3151 Luci#2A5) were seededin 50 μl assay medium in 96-well white assay plates (10,000 cells/well)and grown for 24 h+20 min at 37° C., 7.5% CO2 and 95% rH before additionof the samples. Each 96-well assay plate comprised a total number of 3different negative controls (heat inactivated serum, serum with andwithout IMAB362 and serum with an isotype control antibody (Rituximab))and a positive control of healthy human serum pool (lot #31032011) with500 ng/ml IMAB362. An additional positive control was generated at theend of the reaction by addition of 0.8% Triton X100 to a second mediumcontrol well causing total lysis. One of the 96-well assay platescomprised a functional positive control generated by 7 serial 3.16 folddilutions of IMAB362 (10 000-31.8 ng/ml). This control resulted in asigmoid dose-dependent lysis of target cells. All samples were prepared(200 μl each) at the same time in a 96 well deepwell dilution plate.Samples were taken 3 times from each well by reverse pipetting togenerate the triplicates in the assay plates. After addition of 50 μl ofeach test and control item to the assay plates, plates were incubatedfor 80+5 min at 37° C., 7.5% CO2 und 95% rH.

To each well 10 μl PBS was added, except in the Triton-Lysis controlwells. To each Triton-Lysis control well, 10 μl 0.8% Triton/PBS solutionwas added. Luciferin substrate solution was prepared (6114 μl Aquabidest, 2496 μl HEPES (1M), 1998 μl 1×DPBS, 4992 μl D-LuciferinStocksolution (12 mg/ml)). To each well 50 μl Luciferin substratesolution was added. Plates were incubated at 37° C., 7.5% CO2 und 95% rHfor 45 min. Plates will be measured in a microplate reader.

-   -   Complement-dependent lysis was calculated using formula:

Specific lysis(%)=100−[(RLUsample−RLUtriton)/(RLUHSCM−RLUtriton)×100]

Modifications for testing the pancreatic cancer cell lines:

-   -   Pancreatic cancer cells were transfected with luciferase RNA        using optimized conditions. For each cell line tested, 1.5×104        cells were seeded per well.    -   Since most pancreatic cancer cell lines are difficult to detach        and to singularize, trypsin was used on day 1.    -   Pancreatic cancer cells in assay plates were cultured at 37° C.,        5% CO2.    -   CDC assays with chemotherapeutic agents pretreated cells was        done with following IMAB362 or as isotype control antibody        ch78H11 1H6 antibody concentrations: 640000, 160000, 40000,        10000, 2500, 625, 156 and 39 ng/ml.

11. Inhibition of Proliferation

To analyze dose-response curves of each chemotherapeutic agent, aproliferation assay was performed.

TABLE 6 Pancreas cancer cell lines to analyze efficacy of gemcitabine oroxaliplatin Inhibition of proliferation after applying gemcitabine oroxaliplatin in different concentrations for each pancreas cancer cellline was analyzed. cell line cells seeded/well BxPC3-LVT 5000 BxPC3 5000Panc05.04 5000 BxPC3-LVT 5000 CAPAN1-LVT 5000 DANG 2000 MiaPaCa-2-LVT7000 Patu8988S 10000 Patu8988Sp3151#6 15000

Cells were seeded in 96 well plates and after 4-6 hours gemcitabine oroxaliplatin was added in following concentrations: 1000, 500, 250, 100and 20 ng/ml. The proliferation assay was incubated for 4 days at 37° C.and 5% CO2. 50 μl XTT complete reagent (50 parts XTT+1 part coupl.reagent mixed) was added and incubated at 37° C. Measurement ofabsorbance (cells plus supernatant) was done with the Tecan Safire after3 h and 4 h. Inhibition of proliferation was calculated compared tomedium values set as 100%. EC₅₀ values for gemcitabine and oxaliplatinwere calculated in the GraphPad Prism program.

12. Cultivation of Pancreas Cancer Cell Lines with ChemotherapeuticDrugs for ADCC or CDC

For DANG 4 to 6E+06 cells were seeded and cultivated for 2 days inmedium or medium+1 ng/ml gemcitabine or 1 ng/ml gemcitabine+10 ng/mloxaliplatin. 1-1.4E+07 Patu8988S were seeded and cultivated without orwith 10 ng/ml gemcitabine or 10 ng/ml gemcitabine in combination withoxaliplatin 100 ng/ml.

On the day ADCC started, the protocol described above was followed andcell surface expression of CLDN18 was determined in FACS analysis asdescribed above.

13. Cell Cycle Analyses

Cells were plated in six-well plates, and 5-6 hours laterchemotherapeutic agents were added for either 24 h 48 h or 3 days. Cellsfloating in the medium were combined with the adherent cell layer, whichwas trypsinized. Cells were washed. Either cell cycle analysis wasstarted directly or cell surface staining was done before as describedabove. Cells are resuspended in 1 ml PBS and added to 3 ml 4% PFA. After15 min fixation of cells at room temperature cells are pelleted andwashed. For RNAse treatment cells were resuspended in 200 μl RNAse(10000 U/ml) plus 0.05% Triton X-100 and incubated for 30 min at 37° C.1 ml PBS was added and samples were centrifuged and resuspended in 200μl PBS/PJ 50 μg/ml. At least 30 min later samples were ready to beanalyzed by flow cytometry. Cell cycle phase distribution was determinedusing FlowJo software to analyze DNA content histograms.

14. Apoptose Assay

Following the indicated treatments, apoptosis was measured by annexin Vbinding (detection kit I) or by a DNA fragmentation assay (Apo-Direct)as recommended by the manufacturer (PharMingen, San Diego, Calif.).Briefly, cells floating in the supernatant were combined with theadherent fraction, which was trypsinized and then washed. An aliquot of5E+05 cells was incubated with annexin V-APC and PI for 15 min at roomtemperature in the dark. Cells were immediately analyzed by flowcytometry. Viable cells exclude both annexin V-APC and PI. Earlyapoptotic cells are annexin V-APC-positive and PI-negative, whereascells that are no longer viable due to apoptotic or necrotic cell deathare positively stained by both annexin V and PI. Percentage of stainedcells in each quadrant was quantified using FlowJo software (BDBiosciences, Franklin Lakes, N.J.).

The apoptotic assay based on DNA fragmentation was performed as follows.Treated cells (adherent and floating) were fixed in 70% icecold EtOHovernight. After washing, 10⁶ fixed cells were incubated with terminaldeoxynucleotidyl transferase enzyme (TdT) and FITC-dUTP for 90 min at37° C. to label DNA breaks. Cells were rinsed, incubated in RNaseA/propidium iodide in the dark for 30 min at room temperature to staintotal DNA, then analyzed by flowcytometry. Cell doublets and clumps wereeliminated from the analysis by gating.

15. In Vivo Studies

All in vivo experiments were carried out in compliance with the nationalregulations and ethical guidelines for experimental animal studies.

15.1 Treatment of Xenografts

Xenograft tumors were inoculated by subcutaneous injection of tumorcells in 200 μl PBS into the flanks of female Hsd:AthymicNude-Foxn1^(nu) mice. Tumor bearing mice were treated with 0 μg, 200 μg,400 μg or 800 μg antibody injected i.v. weekly or alternating i.v./i.p.semi-weekly. Chemotherapeutic agents were applied i.p. weekly orsemi-weekly. Tumor sizes and animal health were monitored semi-weekly.At the end of chemotherapy treatment, antibody applications werecontinued until tumors reached a volume of >1400 mm³ or until tumorsbecame ulcerous. Tumor samples were cryo conserved or fixed in 4%formalin for subsequent analysis.

15.2 Metastasis Assay

Different pancreatic cancer cell lines were first analyzed for theirability to form metastasis after i.v. application of the cells in nudemice. For these engraftment analyses a group of 5-10 mice were injectedwith 1×10⁶ and/or 2×10⁶ cells and single mice were sacrificed atdifferent time points to find the time point of metastasis engraftmentand growth.

Metastasis treatments were performed with 10-12 Hsd:AthymicNude-Foxn1^(nu) mice per treatment group. They were injected with 2×10⁶cells (Patu8988S or Suit2-LVT) intravenously. All mice were sacrificedat the same time point, as soon as the first symptoms of metastasisdisease appeared (loss of weight, weakness, shortness of breath), or thefirst mouse died.

Preparation of Tissue:

For engraftment studies mice were sacrificed at different time points,or as soon as mice showed clear physiological signs of metastaticdisease (loss of weight, weakness, shortness of breath). All theirorgans were macroscopically analyzed for metastasis. Only for Patu8988Sand Suit-2 cells, lungs and lungs/livers displayed macroscopicallyvisible metastasis, respectively. These organs were dissected into 4equal pieces, two (lung: upper right and lower left lobe), which werestored for genomic DNA isolation. The other two pieces were formalinfixed and stored for IHC analysis (FIG. 2).

Preparation of Genomic DNA and Q-PCR Strategy:

Genomic DNA was extracted from lung or liver tissue. As controls,genomic DNA was also isolated from human pancreatic cancer cellsPatu8988S as well as of a non-injected negative control mouse.

The Q-PCR strategy is based on the amplification of human DNA present inthe metastases. The relative detection level of human DNA in the mouselung sample correlates directly with the amount and/or size of themetastases. Since this method is biased by the fact that the metastasesdo not spread evenly in the lung and sometimes one lobe is more affectedthan the other, two different regions of the lungs were mixed in one DNApreparation (FIG. 2).

The Q-PCR reaction was performed with primer pair #58615′-GGGATAATTTCAGCTGACTAAACAG-3′ and #58625′-TTCCGTTTAGTTAGGTGCAGTTATC-3′ specifically amplifying thealpha-satellite DNA present in human chromosome 17, but not in mouseDNA. To generate a standard curve and as positive control, Patu8988S DNAwas mixed with mouse DNA and 5 fold dilutions were prepared, resultingin 100%, 20%, 4%, 0.8%, 0.16%, 0.032% and 0.0064% human DNA in mouseDNA. The curve was used to calculate (linear regression) the amount ofhuman metastasis DNA present in mouse lung tissue. Q-PCR reactions wereperformed in 50 μl final volume comprised of 20 μl (200 ng) mouse lungDNA, 25 μl Sybr Green (Qiagen), 1.6 μl sense primer (10 μM) and 1.6 μlanti-sense primer and 1.8 μl H₂O.

Example 2 CLDN18.2 Expression in Normal and Neoplastic Human PancreasTissues

To analyze the expression level and pattern of CLDN18.2 in normal andpancreatic tumor tissues histological staining of FFPE sections wascarried out with two murine monoclonal antibody reagents (FIG. 3).

Exploratory pilot experiments were performed by using the prototypeantibody 35-22A on tissue microarrays (TMAs). A major disadvantage ofTMAs is the variable quality of the spotted tissues and the small sizeand thus non-representative character of the samples. This together withthe not fully optimized staining protocol may have resulted in anunderestimation of positive cases.

The main experiments were performed with the antibody 43-14A. Thesestainings were conducted on tissue sections, which (compared to theTMAs) were larger and pre-assessed for presence of tumor cells.

The precancerous lesions, which origin from the pancreas ducts can beranked according to the international pancreas intraepithelial neoplasia(PanIN) system (PanIN-1A, -1B, -2, -3 subtype). PanIN-1 lesions (FIG.4A) are flat, composed of tall columnar cells with basally locatednuclei and abundant supranuclear mucin. The nuclei are small and roundto oval in shape and are oriented perpendicular to the basementmembrane. There is histological overlap between non-neoplastic flathyperplastic lesions and flat neoplastic lesions without atypia.

The lesions of the subtype PanIN-1B have a papillary, micropapillary orbasally pseudostratified architecture and are otherwise identical toPanIN-1A. (Hruban et al. Am J Surg Pathol. 2001 May; 25(5):579-86.)

PanIN-2 lesions (FIG. 4B) are flat or papillary, have typical nuclearabnormalities, including some loss of polarity, nuclear crowding,enlarged nuclei, pseudo-stratification and hyperchromatism. Mitoses arerare, but when present are non-luminal (not apical) and not atypical.(Hruban et al. Am J Surg Pathol. 2001 May; 25(5):579-86.) PanIN-3lesions (FIG. 4C) are usually papillary or micropapillary, however, theymay rarely be flat. True cribriforming, budding off of small clusters ofepithelial cells into the lumen and luminal necroses suggest thediagnosis of PanIN-3. Lesions are characterized by a loss of nuclearpolarity, dystrophic goblet cells (goblet cells with nuclei orientedtowards the lumen and mucinous cytoplasm oriented toward the basementmembrane), mitoses which may occasionally be abnormal, nuclearirregularities and prominent (macro) nucleoli. (Hruban et al. Am J SurgPathol. 2001 May; 25(5):579-86.)

The expression of CLDN18.2 in precancerous tissues was analyzed with the43-14A antibody using tissue samples of various sources.

CLDN18.2 was detected frequently in PanIN structures of the subtypesPanIN-1, -2 and -3 demonstrating an early expression of CLDN18.2 inprecancerous lesions (FIG. 4), which is conserved in the later stages.In contrast, no expression was observed in normal pancreas tissuesamples including the ductal structures of the pancreas.

In conclusion, CLDN18.2 is an early marker of beginning malignanthistological changes in the pancreatic ducts.

Two studies were performed to assess expression of CLDN18.2 in primarypancreatic cancer.

For the pilot study several TMAs with a total of 141 primary pancreaticcancer cases were stained with the monoclonal CLDNA18.2 specificantibody 35-22A. The overall quality of the analyzed TMA wasunsatisfying. Many spots were partially lost during the retrieval andinhomogenous counterstaining with haematoxylin was suggestive forsuboptimal tissue processing of FFPE tissues.

Overall >48.9% of the stained cases were positive for CLDN18.2,including 49.2% (65/132) ductal adenocarcinomas, 50% (½) acinic cellcarcinoma and 3 of 7 neuroendocrine carcinomas (Table 7). The tumor cellmembrane was stained without any background on other cell types (FIG.6).

Moreover, we observed a correlation between CLDN18.2 expressionintensity and the fraction of stained tumor cells within the tumor(Table 8, FIG. 5).

TABLE 7 Pilot study: Number of CLDN18.2 positive cases divided in thepancreas cancer subtypes. Tissues were stained using the monoclonal,murine 35-22A (0.2 μg/ml) antibody and reviewed for CLDN18.2 positivetumor cells. positive staining fraction ≧1% intensity primary pancreasCA total [%] ≧2+ [%] total 141 69 [48.9] 62 [43.9] ductal adenocarcinoma(PDAC) 132 65 [49.2] 58 [44.3] acinic cell carcinoma 2  1 [50.0]  1[50.0] neuroendocrine carcinoma 7  3 [42.8]  3 [42.8]

TABLE 8 Pilot study: Correlation between CLDN18.2 signal intensity andamount of positive tumor cells for the analyzed pancreas primary tumors.Percentage of positive primary tumor cases correlated to the stainingintensity. The cases were grouped in six fractions depending on theamount of positive tumor cells for a better visualization. signalIntensity % of pos. cells + ++ +++ Total any positivity 7 24 38 69  1-9%3 [42.8] 6 [25.0] 1 [2.6] 10 [14.5] 10-39% 2 [28.6] 6 [25.0]  5 [13.2]13 [18.8] 40-49% 0  0 1 [2.6] 1 [1.4] 50-59% 0 3 [12.5] 2 [5.3] 5 [7.3]60-69% 0 2 [8.3]   5 [13.2]  7 [10.2] 70-100% 2 [28.6] 7 [29.2] 24[63.1] 33 [47.8]

TABLE 9 Pilot study: Grading of the CLDN18.2 positive tumor cases. Thegrading of the tumor cells describes the cell appearance and the levelof cell differentiation. Whereat grade 1 describes well differentiatedcells; grade 2 moderately differentiated cells and grade 3 poordifferentiated. positive total fraction staining intensity grade cases≧1% [%] ≧2+ [%] 1 15 13 [86.7] 12 [80.0] 2 71 39 [54.9] 36 [50.7] 3 35 9 [25.7]  7 [20.0]

A second study was conducted with an optimized staining protocol withthe highly sensitive antibody 43-14A using quality controlled tissuesections.

TABLE 10 Main study - number of CLDN18.2 positive cases grouped inpancreas cancer subtypes. Tissues were stained using the murine,monoclonal 43-14A (0.2 μg/ml) antibody and reviewed for CLDN18.2positive tumor cells. positive staining total fraction intensity primaryCA cases ≧1% [%] ≧2+ [%] total 61 40 [65.6] 39 [63.9] ductaladenocarcinoma (PDAC) 42 38 [90.5] 37 [88.1] acinic cell carcinoma 1 0 0neuroendocrine 18  2 [11.1]  2 [11.1] Table A primary cholangio CA totalpositive % pos. cholangio carcinoma 15 9 60 Table B

In total 42 primary ductal pancreatic cancer samples were analyzed.About 90% (38 of 42 cases) of these were positive for CLDN18.2 (Table10), most of which (>60%) showed a strong signal intensity of +++ (FIG.7, Table 11). Also here a correlation between the CLDN18.2 expressionlevel and the fraction of positive tumor cells was observed. Most of theanalyzed cases (62%) were grade 3 tumors (Table 12).

TABLE 11 Main study: Correlation between CLDN18.2 signal intensity andamount of positive tumor cells for the analyzed pancreas primary tumors.Percentage of positive primary tumor cases correlated to the stainingintensity. The cases were grouped in six fractions depending on theamount of positive tumor cells for a better visualization. signalintensity % of pos. cells + ++ +++ Total any positivity 1 13 26 40  1-9%1 2 [15.4] 3 [11.5] 6 [15.0] 10-39% 0 3 [23.0] 4 [15.4] 7 [17.5] 40-49%0 2 [15.4] 2 [7.7]  4 [10.0] 50-59% 0 2 [15.4] 4 [15.4] 6 [15.0] 60-69%0 2 [15.4] 1 [3.8]  3 [7.5]  70-100% 0 2 [15.4] 12 [46.2]  14 [35.0] 

TABLE 12 Main study - Grading of the CLDN18.2 positive tumor cases. Formost of the analyzed tumor cases a grading done by the correspondingpathologist was available. The grading of the tumor cells measures thecell appearance and the level of cell differentiation. Whereat grade 1describes well differentiated cells; grade 2 moderately differentiatedcells and grade 3 poorly differentiated. positive staining totalfraction intensity grade cases ≧1% [%] ≧2+ [%] 1 7  1 [14.3]  1 [14.3] 225 15 [60.0] 15 [60.0] 3 28 24 [85.7] 23 [82.1]

Pancreas cancer is diagnosed in most patients in an advanced stage.Patients tumors have already metastasized in lymph nodes and otherorgans, in particular into the liver. In the main study 79 FFPE tissuesamples of lymph node and liver metastases of pancreatic cancer wereanalyzed in an immunohistochemical assay, using the CLDN18.2 specific43-14A antibody.

70.5% of the lymph node metastases (31/44 cases) and 68.6% of thedistant liver metastases (24/35 cases) demonstrated clear tumor cellstaining for CLDN18.2 (Table 13). The staining pattern of the positivetumor cells was membranous, in some cases with additional weakercytoplasmic signals (FIG. 9). In accordance with the results of theprimary tumor analysis, a correlation was found between the CLDN18.2expression level and the fraction of CLDN18.2 positive tumor cells inthe metastatic samples (FIG. 8).

No correlation was found between grading of the analyzed tumors andexpression level of CLDN18.2 or fraction of positive tumor cells.

TABLE 13 Number of CLDN18.2 positive metastases cases grouped by targetorgan. Tissues were stained using the monoclonal, murine 43-14A (0.2μg/ml) antibody and reviewed for CLDN18.2 positive tumor cells. positivestaining fraction intensity metastasis total ≧1% [%] ≧2+ [%] total 79 55[69.6] 52 [65.8] pancreas to lymph node 44 31 [70.5] 28 [63.6] pancreasto liver 35 24 [68.6] 24 [68.6]

To test whether the CLDN18.2 expression of positive primary tumor casesis conserved in metastases of the same patient, matched primarycancer/lymph node metastases doublets were screened using antibody43-14A.

TABLE 14 CLDN18.2 expression in matched pancreas primary and lymph nodemetastatic tumor samples - Matched samples of primary adenocarcinoma andlymph node (LN) metastasis were analyzed for the expression of CLDN18.2in tumor cells. Patient ID Primary TU LN met. H/2011/775 3S & 8D +++(15%) +++ (25%) H/2011/2247 7C & 7G +++ (10%) ++ (25%) H/2011/12675 4B &4L +++ (10%) ++ (5)% H/2010/15941 6SS & 8H +++ (90%) +++ (90%)H/2010/2986 2E & 2G +++ (5%) +++ (40%) H/2010/6709 9B &9 F +++ (70%) +++(10%) H/2010/11569 5A & 5G +++ (80%) ++ (5%) H/2008/380 4A & 4G +++(60%) +++ (90%) H/2009/13538 5B & 5E + (20%) + (10%) H/2009/11847 7C &7F +++ (15%) ++ (15%) H/2009/23108 4A & 4J +++ (5%) +++ (70%)H/2009/4917 8E & 8S +++ (70%) +++ (25%) H/2009/214183C & 3L +++ (55%) ++(70%) H/2009/20336 2D & 2F +++ (80%) +++ (90%) H/2009/17768 2C & 2E ++(35%) ++ (20%) H/2008/13194 3A & 3J +++ (35%) +++ (10%) H/2008/13074 3B& 3C +++ (50%) +++ (50%) H/2008/12082 6D & 6G +++ (1%) − H/2008/11178 5B& 1SS +++ (80%) +++ (90%) H/2008/28150 4B& 4C ++ (15%) ++ (30%)H/2007/5478 4A & 4J +++ (90%) +++ (90%) H/2007/6216 3B & 3C +++ (50%)+++ (60%) H/2007/9047 1B & 1J +++ (70%) +++ (35%) H/2007/13983 6C & 6J++ (15%) ++ (1%) H/2007/14400 6C & 6J − − H/2006/5616 3J & 3D +++ (35%)+++ (5%) H/2006/9779 3D & 3K + (5%) + (15%)

In 25 (92.5%) of the 27 analyzed paired cases both primary tumor andlymph node pairs were positive for CLDN18.2. In a single case bothtissues were negative and in one other case the primary tumor waspositive for CLDN18.2, whereas the metastasis was negative.

In 21 of 26 (80.7%) positive tested doublets the signal intensity of theprimary tumor and metastatic tumor cells was identical. In 5 cases thesignal intensity declined from +++ to ++. In 11 of 25 (44%) pairedtissues the number of positive tumor cells was lower in the metastasesas compared to the primary tumor (Table 14).

In summary, the CLDN18.2 expression appears to be conserved when primarytumor cells advance to the metastatic stage. The overall intensity andthe fraction of positive tumor cells in lymph node metastases was onlyslightly lower as compared to the primary tumor (FIG. 10).

For a small number of patients tissue samples derived from the primarytumor, the lymph node metastasis and the liver metastasis wereavailable. These matched triplets were stained, to test the conservationof CLDN18.2 expression in distant metastases. Six matched triplets wereanalyzed with antibody 43-14A.

TABLE 15 CLDN18.2 expression in matched pancreas primary and metastatictumor samples - Matched samples of primary adenocarcinoma, livermetastasis and lymph node (LN) metastasis were analyzed for theexpression of CLDN18.2 in tumor cells. primary lymph node liver patientID tumor metastasis metastasis H/2011/17191-VA-VI-ISS +++ (60%) +++(70%) +++ (1%) H/2010/14296 XA +++ (5-10%) − − H/2010/4157 VII A ++(60%) ++ (30%) +++ (90%) H/2009/23598 VII B +++ (40%) − −H/2011/3590-III SS-VIIC-I ++ (20%) − − H/2008/10701 +++ (90%) +++ (100%)+++ (90%)

In 3 of 6 triplets, all three tissues specimen were comparable withregard to their positivity score for CLDN18.2 (FIG. 11). In three casesa fraction of the tumor cells was CLDN18.2 positive in the primarylesion, whereas the metastatic lesions showed no CLDN18.2 staining(Table 15).

Example 3 Target Expression in Human Pancreatic Cancer Cell Lines Usedfor In Vitro and in Vivo Models and Pancreatic Cancer Models

Source of Cell Lines

A primary aim of this pre-clinical evaluation study was to analyze theinhibitory effects of IMAB362 treatment in suitable model systems. Toidentify CLDN18.2-positive cell lines that can be used for in vitro andin vivo characterization of IMAB362 effects, a set of 26 commerciallyavailable pancreatic cancer cell lines was screened for CLDN18.2expression and characterized in detail. A cell bank for experimental usewas prepared immediately upon arrival for each of the cell lines. Theywere derived from primary pancreatic adenocarcinomas (10 of which 6mucinous adenocarcinomas), primary carcinomas (4), pancreaticadenocarcinomas metastases into liver (5) or spleen (1), or isolatedfrom ascites (5) (see Table 16). Several of these cell lines (8) werelentivirally transduced to express CLDN18.2.

TABLE 16 Published origin and cellular characteristics of pancreas celllines. Human In vivo growth: Cell line Supplier¹ Origin² s.c. tumorsmetastasis Additional information Cellular products³ References AsPC1ATCC AS, yes Only after Orthotopic: metastasis in gut, kidney CEA,pancreas Chen W H et al. In Vitro: ADCA orthotopic and peritoneum.cancer associated 18: 24-34 (1982) transplantation Intrasplenic: Livermetastasis antigen, pancreas Tan and Chu et al., or intrasplenicspecific antigen, 1985 Tumor Biol 6: 89-98 injection in mucin SCID andNOG BxPC3 ATCC PT, yes Only after Negative for cystic fibrosis CEA,pancreas Tan M H et al. Cancer ADCA intrasplenic transmembraneconductance cancer associated Invest. 4: 15-23 (1986) injection NOG,regulator (CFTR) negative. antigen, pancreas Suemizu et al. Int. J. notin Nu/Nu, specific antigen, Oncol. 31: 741-2007 NOD/SCID mucin BxPC3ECACC PT, yes Tumorigenic in nude mice Mucin, CEA, Tan M H et al. Cancer(ECACC) ADCA generating moderately well to poorly pancreas Invest. 4:15-23 (1986) differentiated tumors comparable to cancer associatedprimary adenocarcinoma. antigen, pancreas specific antigen CAPAN1 DSMZLM, yes After Positive for cystic fibrosis Fogh et al. J. Natl. D-ADCAintrasplenic transmembrane conductance Cancer Inst. 58: 209-214injection in regulator (CFTR). Resistant to 5-FU (1977). NOG Suemizu etal. Int. J. Oncol. 31:741- 2007 CFPAC1 ATCC LM, yes After Expressproduct of the CF gene (cytic CEA, pancreatic Schoumacher R A et al. D-orthotopic fibrosis). Cells have the most oncofetal antigen, Proc Natl.Acad Sci. USA ADCA, transplantation common form of the CF mutation.adenocarcinoma 87: 4012-4016 (1990). cystic in Nu/Nu No effect of cAMPagonists, adenyl associated Lee et al., Oncogene fibrosis cyclasestimulators or antigen, Ca19-9, 2010, 29: 56-67. phosphodiesteraseinhibitors. epithelial keratins Thayer et al., Nature Respond to Ca++ionophores. 2003 425: 851 Capecitabine and cyclopamine sensitive DANGDSMZ CA yes No Not published HPAFII ATCC AS, yes Low metastatic IL22-Rpositive (inhibits NK cell Mucin1 and Kim Y W et al. Pancreas ADCA afteraction via IL10 and TGF-(31) Mucin 4 4: 353-362, 1989. Curd orthotopicet al., clin. Exp. transplantation Immunol. 168 (2012) in SCID HPAC ATCCADCA yes Low metastatic Derived from pancreas head. Tumors EGF,functional Gower W J et al. In vitro after in nude mice histologicallysimilar to glucocorticoid Cell Dev Blot 30A: 151- orthotopic tumor oforigin. Growth stimulated receptor, keratin, 161(1994) transplantationwith insulin, IGF-I, EGF and TGFα. pancreatic ductal in SCID Growthsuppressed by epithelium marker dexamethasone and glucocotricoids.(DU-PAN-2), antigens (HMFG1, AUA1) tumor-associated antigens (CEA,CA-125, CA19-9) HUP-T3 DSMZ AS, CA From poorly differentiated ADCA Smallamounts Nishimura et al. Int. J. of CEA, Pancreatol 13: 31-41 TGFβ2(1993) HUP-T4 DSMZ AS, CA From well-differentiated Large amountsNishimura et al. Int. J. papillotubular ADCA of CEA Pancreatol 13: 31-41and CA19-9, (1993) Schlingensiepen TGFβ2 et al. Antisense Pharma GmbHKCI-MOH DSMZ PT- Yes (SCID) Derived from pancreas head. CytokeratinsMohammad et al., ADCA Moderately differentiated, tubular. Pancreas 16:19-25 (1998) KP-2 JCRB D- yes (minimally) Moderately differentiated.Ikeda et al., J Cancer ADCA Transplantable to nude mice Res. 81: 987-993(1990) histologically similar to tumor of origin. Minimally metastatic.KP-4 JCRB D-CA yes highly Parathyroid Nishi et al., Int. J.hormone-related Oncol. 5: 33-39 (1994) peptide (PTHrP) MiaPaCa2 JCRB PT-CA yes no Undifferentiated, sensitive to Yunis et al., Int. J.asparaginase. cancer 19: 218-235 (1977) Panc01 ATCC PT, D-E- Yes Yes,after Suemizu et al. Int. J. CA (NOD/SCID, intrasplenic Oncol. 31:741-2007 NOG) injection in NOG Panc02.03 ATCC PT, Yes Derived frompancreas head. K-ras Cytokeratins Jaffee et al., cancer J. ADCA(nude/SCID) oncogene mutation Sci. Am. 4: 194-203 (1998) Panc03.27 ATCCPT, Yes Derived from pancreas head. Wild- Cytokeratins Jaffee et al.,Cancer J. ADCA (nude/SCID) type K-ras Sci. Am. 4: 194-203 (1998)Panc04.03 ATCC PT, Yes Derived from pancreas head. K-ras CytokeratinsJaffee et al., Cancer J. ADCA (nude/SCID) oncogene mutation Sci. Am. 4:194-203 (1998) Panc05.04 ATCC PT, ony if Derived from pancreas head.K-ras Cytokeratins Jaffee et al., Cancer J. ADCA transplanted oncogenemutation. S.c. tumors Sci. Am. 4: 194-203 in matrigel sensitive forcyclopamine. (1998). Thayer et al., Nature 2003 425: 851 Patu8902 DSMZPT, yes yes Grade II, Secretes protinases Elsasser et al., D-ADCA andcathepsin B, Virchows Arch B Cell expression Pathol Incl Mol of TGFβ2Pathol, 64: 1993, 201 Patu8988S ATCC LM, yes Lung only Undifferentiatedsolid tumors in Cytokeratins, Elsasser et al., ADCA mice. no mucinVirchows Arch B Cell Pathol Incl Mol Pathol 61: 295-306 (1992) Patu8988TATCC LM, yes no Differentiated tumors in mice with High mucin Elsasseret al., ADCA tubular structures (sister cell line of secretion, VirchowsArch B Cell Patu8988S), non-metastatic in mice. Cytokeratins Pathol InclMol Pathol 61: 295-306 (1992) Suit2 HSRRB LM, T- yes highly Moderatelydifferentiated tubular, CEA, CA19-9 Ywamura T. et al ADCAepithelial-like. Highly metastatic in nude mice Su86.86 ATCC LM, YesCells can be lysed by LAK cells in CEA Drucker B J et al., In vitroD-ADCA (orthothopic) presence of IL-2 but not by NK cells Cell Dev Biol.24: 1179- 1187, 1988 SW1990 ATCC SM, Yes Grade II, derived from exocrineMucin, CEA Kyriazis A P et al., Cancer ADCA pancreas, epithelial, tumorsin nude Res. 43: 4393-4401 (1983) mice resemble original tumor, ductalmorphology YPAC DSMZ AS, CA Yes Yes Forms tumors in nude mice withSecrete Yamada et al., Int J functional characteristics of originalinflammatory Cancer, 76: 1998, 141, tumor. cytokines, IL1α (autocrine),IL6, IL8 ¹ATCC: American Type Culture Collection; HSRRB: Health ScienceResearch Resources Bank, DSMZ: Deutsche Sammlung von Mikroorganismen andZellkulturen. ²PT: primary tumor, AS: ascites, LM: liver metastasis, SM:spleen metastasis, ADCA: adenocarcioma, D: ductal, T: tubular, E:epitheloid, CA: pancreas carcinoma ³CEA: carcinoembryonic antigen,CA:carbohydrate antigen, IL: interleukin

CLDN18.2 Transcript Expression in Human Pancreatic Cancer Cell Lines

To identify CLDN18.2-expressing pancreas cell lines, transcript levelswere determined with quantitative real-time PCR (RT-PCR) using a forwardprimer binding to exon 1 of CLDN18.2 and a reverse primer binding toexon 3 of CLDN18. The endogenously CLDN18.2 expressing human gastriccarcinoma cell line KATO-III and the CLDN18.2 negative breast cancercell line SKBR-3 were included as positive and negative controls,respectively. The RT-PCR revealed clear endogenous CLDN18.2 expressionin the pancreatic cancer cell lines DANG, Panc03.27, Panc05.04,Patu8988S and YAPC with relative levels exceeding 1×10⁵. Interestingly,Patu8988S cells showed CLDN18.2 expression levels (˜1×10⁸) comparable tostomach CA KATO-III cells (FIG. 12A). In conclusion, we detected robustCLDN18.2 expression in 5 out of 22 pancreatic cancer cell lines.

In addition to the endogenous cell lines, the LVT cell lines ectopicallyexpressing CLDN18.2 were analyzed on the transcript level (FIG. 12A).For 6 out of 8 LVT cell lines, relative CLDN18.2 expression levels ofmore than 1×10⁸ were detected. Only in HAPC-LVT and Suit2-LVT cells, theexpression level was above 1×10⁵.

We investigated if CLDN18.2 expression is stable during in vitrocultivation. Patu8988S, Panc05.04 cells and the lentivirally transducedcell lines Suit2-LVT, MiaPaCa2-LVT and Patu8902-LVT were passaged up to15 times and CLDN18.2 transcript was analyzed (FIG. 12B-D). We observedloss of CLDN18.2 expression in both endogenous and transduced cells witha higher passage number. Loss of expression was highest in thetransduced cells. Therefore, early passages were used, wherever possiblefor the in vitro experiments and expression of CLDN18.2 in tumorxenografts was verified in the below engraftment experiments.

CLDN18.2 Protein Expression in Human Pancreatic Cancer Cell Lines

Detection of CLDN18.2 in Total Cell Lysates

In addition to the transcript analyses, the expression of CLDN18.2 wasanalyzed on the protein level by Western blot and IF. For Western blotanalysis cell lysates of the 26 pancreatic cancer cell lines wereinvestigated by western blotting (WB) using the CLDN18 specific antibodyanti-Claudinl 8 (C-term). Lysates of SKBR-3 cells were again used asnegative control, whereas lysates of HEK293 cells stably transfectedwith CLDN18.2 (HEK293-p740) were used as positive control. Here, wedetected high protein expression in Patu8988S, DANG and Panc05.04 cells,confirming the RNA data. Faint bands were detectable in Panc03.27 andBxPC3 cell lysates. YAPC cells, which were identified positive on theRNA level showed a faint band of smaller size in western blots. Allother cell lines were negative (FIG. 13).

Cellular Expression of CLDN18 in Pancreatic Cancer Cells

To obtain supportive protein expression data, pancreatic carcinoma celllines were investigated by immunofluorescence (IF) after fixation andpermeabilization of the cells and using antibody 35-22A for detection.IF analyses confirmed previous RNA and protein data showing that mostpancreatic cancer cell lines are negative for CLDN18.2 staining (FIG.14). In a few cell lines (like AsPC1, DANG, HUP-T3, HUP-T4, Panc01)nuclear dots were observed, which most likely represent stainingartifacts. DANG, Panc03.27 and BxPC3 cells that were identified tofeature low CLDN18.2 on the RNA and/or protein level, were negative inthe IF analysis, which has a lower detection sensitivity. In contrast,membranes and cytoplasm of Panc05.04, Patu8988S and KATO-III gastriccarcinonoma control cells stained strongly positive for CLDN18.2.Staining intensity was different for each cell and also negative cellswere detected within the population (FIGS. 14J and N). In the LVT celllines we found strong membrane staining of more than 80% of all cells.

Confirmation of CLDN18.2 Expression in Pancreatic Cancer Cells

To confirm expression of CLDN18.2 and to assess amount of this target onthe cell surface the endogenous cell lines Panc05.04 and Patu8988S aswell as in the LVT cell lines were stained with IMAB362 using a nativestaining protocol. Although staining of Patu8988S, Panc05.04 and theKATO-III gastric cancer control cells with IMAB362 was less intense andthe percentage of positive cells was reduced compared to staining cellswith 35-22A (FIG. 16A-F), the IF analysis confirmed that CLDN18.2 isexpressed on the surface of pancreatic cancer cells. For the 8 LVTpancreatic cancer cell lines ectopically expressing CLDN18.2, clearmembrane staining was observed on almost all cells (as shown for 6 LVTcell lines in FIG. 16G-L).

In conclusion, the CLDN18.2 expression analyses resulted inidentification of endogenously expressing pancreatic cancer cell linesPanc05.04 and Patu8988S and all 8 lentivirally transduced cell linesBxPC3-LVT, CAPAN1-LVT, DANG-LVT, MiaPaCa-2-LVT, Suit-2-LVT, Patu8902-LVTand YAPC-LVT as suitable CLDN18.2 positive cell model systems.

Development of Pancreatic Cancer Xenograft and Metastasis Models

Engraftment Studies for Identification of Suitable SubcutaneousPancreatic Cancer Tumor Models

A total of 37 engraftment studies with different pancreatic cancer celllines were performed to identify suitable subcutaneous xenograft modelsfor testing in vivo efficacy of 1MAB362. Of all tested cell lines, theBxPC3-LVT, CAPAN1-LVT, MiaPaCa-2-LVT, HPAC-LVT, DANG-LVT and YAPC-LVTcell lines with ectopic CLDN18.2 expression were selected forsubcutaneous xenograft models, showing high engraftment rates andhomogeneous tumor growth. In addition, subcutaneous xenograft modelswith endogenously CLDN18.2 expressing Patu8988S and DANG cell lines wereselected for testing IMAB362 efficacy in vivo. S.c. injection ofPanc05.04 cells did not result in formation of subcutaneous tumors.

TABLE 17 Summary of tested engraftment conditions for development ofs.c. xenograft models for pancreatic cancer. Engraftment Cell line check(EC) Date Experimental set-up Results Comments BxPC3 EC1_BxPC322.03.2010 1e7 cells subcutaneous into the take rate 40%; notrecommended ATCC ATCC left flank of 5 female HsdCpb: median survival 56NMRI-Foxn1nu mice days BxPC3 EC2_BxPC3 23.03.2010 1e7 subcutaneous intothe left take rate 100%; suitable conditions ECACC ECACC flank of 5female HsdCpb: median survival 63 for subcutaneous NMRI-Foxn1nu micedays xenografts BxPC3-LVT EC3_C179 25.03.2011 1e7 cells subcutaneousinto the take rate 90%; suitable conditions left flank of 10 femaleHsdCpb: median survival 64 for subcutaneous NMRI-Foxn1nu mice daysxenografts CAPAN1 EC1_CAPAN1 26.03.2010 1e7 cells subcutaneous into thetake rate 100%; suitable conditions left flank of 5 female HsdCpb:median survival 52 for subcutaneous NMRI-Foxn1nu mice days xenograftsCAPAN1- EC1_C186 09.08.2011 Metastasis assay. 1-2e6 cells in Nometastases in lung not recommended LVT PBS i.v. into 10 female HsdCpb:and liver detected; NMRI-Foxn1nu mice analysis 72 days post tumor cellinjection CAPAN2 EC1_CAPAN2 06.04.2010 1e7 cells subcutaneous into thetake rate 100%; suitable conditions left flank of 5 female HsdCpb:median survival for subcutaneous NMRI-Foxn1nu mice 100 days xenograftsDANG EC3_C2 08.02.2007 1e7 cells subcutaneous into take rate 100%; noadequate the left flank of 3 athymic mice very aggressive tumorconditions for growth; median xenograft model survival 18 days DANGEC4_C2 10.10.2007 5e5-1e7 cells subcutaneous take rate 100%; no adequateinto the left flank of 25 athymic very aggressive tumor conditions formice growth; median xenograft model survival 16 to 23 days DANG EC5_C201.06.2010 2.5e6 cells subcutaneous into take rate 100%, not recommendedsubclone the left flank of 5 female cachexia in tumor due to tumor 1C5F2HsdCpb: NMRI-Foxn1nu mice bearing mice; median cachexia survival 17 daysDANG EC6_C2 10.02.2011 5e4-2e5 cells subcutaneous 5e4 = 40% take rate;not recommended subclone into the left flank of 15 female 1e5, 2e5 =100% take due to tumor 1C5F2 HsdCpb: NMRI-Foxn1nu mice rate; mediansurvival cachexia 26 to 39 days DANG EC7_C2 02.04.2011 2e5 cellssubcutaneous into the take rate 100%; not recommended subclone leftflank of 5 female and 5 cachexia in tumor due to tumor 1C5F2 maleHsdCpb: NMRI-Foxn1nu bearing mice; median cachexia mice; weight kineticssurvival 29 days DANG-LVT EC1_C180 21.03.2011 5e6 cells subcutaneousinto take rate 100%, not recommended the left flank of 10 femalecachexia in tumor due to tumor HsdCpb: NMRI-Foxn1nu mice bearing mice;median cachexia survival 28 days Patu8902 EC1_C197 25.07.2011 1e7 cellssubcutaneous into take rate 100%, fast suitable conditions the leftflank of 5 female tumor growth; for xenograft model HsdCpb: NMRI-Foxn1numice median survival 21 days Patu8902 EC2_C197 25.07.2011 experimentalmetastasis assay. No metastasis in lung not recommended Intravenousinjection of 1-2e6 or liver tissues; 8 mice for metastasis assay cellsinto 10 female HsdCpb: died after tumor cell NMRI-Foxn1nu mice injectionPatu8988S EC1_C178 15.03.2011 1e7 cells subcutaneous into take rate100%, not recommended the left flank of 5 female heterogeneous tumorHsdCpb: NMRI-Foxn1nu mice growth, ulcerating tumors; median survival 55days Patu8988S EC2_C178 05.04.2011 experimental metastasis assay. veryslow metastasis suitable model for Intravenous injection of 1-2e6 model;analysis after lung metastasis cells into 10 female HsdCpb: 70 daysassay NMRI-Foxn1nu mice Patu8988S EC3_C178 10.06.2011 2.5e6-1.5e7 cellsheterogeneous tumor not recommended subcutaneous into the left flankgrowth; slow tumor of 15 female HsdCpb: NMRI- growth; ulcerating Foxn1numice tumors; median survival 95.5 days Patu8988S EC1_C202 11.08.2011 5e6cells subcutaneous into heterogeneous tumor not recommended (recult.)the left flank of 5 female growth; slow tumor HsdCpb: NMRI-Foxn1nu micegrowth; ulcerating tumors; median survival 88 days Patu8988S EC1_C21428.11.2011 5e6 cells subcutaneous into heterogeneous tumor notrecommended subclone the left flank of 5 female growth; slow tumor 17HsdCpb: NMRI-Foxn1nu mice growth; ulcerating tumors; median survival 54days Patu8988S EC1_C215 28.11.2011 5e6 cells subcutaneous into take rate100%, not recommended subclone the left flank of 5 female heterogeneoustumor due to heterogeneous 22 HsdCpb: NMRI-Foxn1nu mice growth; slowtumor and growth; ulcerating ulcerating tumors; tumors; median besttumor growth survival 47 days curves of tested PATU89885 subclonesPatu8988S EC1_C216 28.11.2011 5e6 cells subcutaneous into heterogeneoustumor not recommended subclone the left flank of 5 female growth; slowtumor 30 HsdCpb: NMRI-Foxn1nu mice growth; ulcerating tumors; mediansurvival 35 days Patu8988S EC1_C217 28.11.2011 5e6 cells subcutaneousinto heterogeneous tumor not recommended subclone the left flank of 5female growth; slow tumor 34 HsdCpb: NMRI-Foxn1nu mice growth;ulcerating tumors; median survival 49 days Patu8988S EC1_C218 28.11.20115e6 cells subcutaneous into heterogeneous tumor not recommended subclonethe left flank of 5 female growth; slow tumor 41 HsdCpb: NMRI-Foxn1numice growth; ulcerating tumors; median survival 108 days Patu8988SEC1_C237 06.02.2012 5e6 cells subcutaneous into slow tumor growth;reasonably model subclone the left flank of 5 female bloody cysts, butnot for subcutaneous adM#13 HsdCpb: NMRI-Foxn1nu mice ulcerating; medianxenografts survival 73 days Patu8988S EC1_C238 06.02.2012 5e6 cellssubcutaneous into heterogeneous tumor not recommended subclone the leftflank of 5 female growth; slow tumor adM#19 HsdCpb: NMRI-Foxn1nu micegrowth; ulcerating tumors; median survival 42 days Patu8988S EC1_C23913.02.2012 5e6 cells subcutaneous into heterogeneous tumor notrecommended subclone the left flank of 5 female growth; slow tumor adM#1HsdCpb: NMRI-Foxn1nu mice growth; ulcerating tumors; median survival 77days Patu8988S EC1_C240 13.02.2012 5e6 cells subcutaneous into slowtumor growth; reasonably model subclone the left flank of 5 femalebloody cysts, but not for subcutaneous adM#16 HsdCpb: NMRI-Foxn1nu miceulcerating; median xenografts survival 66 days Patu8988S EC1_C24113.02.2012 5e6 cells subcutaneous into slow tumor growth; reasonablymodel subclone the left flank of 5 female bloody cysts, but not forsubcutaneous adM#9 HsdCpb: NMRI-Foxn1nu mice ulcerating; medianxenografts survival 59 days Suit2 EC1_C196 25.07.2011 1e7 cellssubcutaneous into take rate 100%; fast not recommended the left flank of5 female tumor growth; HsdCpb: NMRI-Foxn1nu mice ulcerating tumors;median survival 35 days Suit2 EC2_C196 25.07.2011 experimentalmetastasis assay. metastases in lung, suitable model for Intravenousinjection of 2exp6 liver and muscles lung metastasis cells into 10female HsdCpb: assay NMRI-Foxn1nu mice Panc02.03 EC1_Panc 12.05.2010 1e7cells subcutaneous into take rate 100%; suitable conditions 02.03 theleft flank of 5 female median survival 54 for subcutaneous HsdCpb:NMRI-Foxn1nu mice days xenograft model Panc03.27 EC2_Panc 12.05.2010 1e7cells subcutaneous into take rate 100%; suitable conditions 03.27 theleft flank of 5 female median survival for subcutaneous HsdCpb:NMRI-Foxn1nu mice 91 days xenograft model Panc04.03 EC3_Panc 17.06.20101e7 cells subcutaneous into take rate 100%; suitable conditions 04.03the left flank of 5 female median survival 39 for subcutaneous HsdCpb:NMRI-Foxn1nu mice days xenograft model Panc05.04 EC4_Panc 18.06.2010 1e7cells subcutaneous into no subcutaneous not recommended 05.04 the leftflank of 5 female tumor growth HsdCpb: NMRI-Foxn1nu mice Panc05.04EC5_Panc 09.05.2011 2e7 cells suspended in RPMI no subcutaneous notrecommended 05.04 subcutaneous into the left flank tumor growth of 5female HsdCpb: NMRI- Foxn1nu mice MiaPaCa2 EC1_C195 25.07.2011 1e7 cellssubcutaneous into the take rate 100%; suitable conditions left flank of5 female HsdCpb: median survival 42 for subcutaneous NMRI-Foxn1nu micedays xenograft model MiaPaCa2- EC1_C219 18.11.2011 5e6 or 1e7 cellssubcutaneous take rate 100%; suitable conditions LVT into the left flankof 10 female median survival 40 for subcutaneous HsdCpb: NMRI-Foxn1numice days xenograft model MiaPaCa2 EC2_C195 25.07.2011 experimentalmetastasis assay. metastases in lung, suitable model for Intravenousinjection of 2e6 liver and lymph nodes lung metastasis cells into 10female HsdCpb: assay NMRI-Foxn1nu mice HPAC EC1_HPAC 19.04.2010 1.5e7cells subcutaneous into take rate 100%; suitable conditions the leftflank of 5 female median survival 29 for subcutaneous HsdCpb:NMRI-Foxn1nu mice days xenograft model YAPC EC1_YAPC 10.05.2010 1e7cells subcutaneous into the take rate 100%, very limited xenograft leftflank of 5 female HsdCpb: aggressive tumor model NMRI-Foxn1nu micegrowth; homogeneous tumor growth; ulcerating tumors; median survival 28days YAPC-LVT EC2_YAPC 10.05.2010 5e5-7.5e6 cells subcutaneous take rate100%, very limited xenograft into the left flank of 20 female aggressivetumor model HsdCpb: NMRI-Foxn1nu mice growth; homogeneous tumor growth;ulcerating tumors; median survival 27 days

Engraftment Studies for Identification of Suitable Metastasis Models

To study effects of IMAB362 on metastasis formation, metastatic cancermodels were established in nude mice. Pancreatic cancer cell lines wereanalyzed for their ability to metastasize upon i.v. application.CAPAN1-LVT, MiaPaCa-2, Patu8988S, Patu8902 and Suit-2 cells wereinjected into tail veins of nude mice as described by Mohanty and Xu2010. To determine the time point of metastasis engraftment and growthrate, the mice were sacrificed at different time points (Table 18).

TABLE 18 Metastasis engraftment analysis of pancreas cancer cell linesOrgans # of # of Mice analyzed on Mice died on isolated Cell line cellsmice day (#) day (#) and stored Metastasis Patu8902 1 × 10⁶  5 — 0(1),1(3) 4(1) n.d. No Patu8902 2 × 10⁶  5 — 0(1), 1(3), 15 (1) n.d. NoPatu8988S 1 × 10⁶  7 34, 51, 59, 66, 70, 86, 108 — Lung/Liver LungPatu8988S 2 × 10⁶  8 34, 41, 51, 59, 66, 70, 86, 108 — Lung/Liver LungSuit-2 2 × 10⁶ 10 36(3), 45(3),52(2), 59(2) — Lung/Liver Lung/LiverCAPAN1- 2 × 10⁶  5 72(1) 0(1), 1(3), n.d. n.d. LVT CAPAN1- 1 × 10⁶  572(4) 2(1) Lung/Liver n.d. LVT MiaPaCa-2 2 × 10⁶ 10 32(2), 52(2), 59(2),66(2), 73(2) — Lung/Liver No

Engraftment analysis of Patu8902 cells and CAPAN1-LVT was not feasible,since most mice died almost immediately. In lungs and livers of 5surviving mice challenged with CAPAN-LVT cells no macroscopicallyvisible metastasis were detected after 72 day. Suit-2 and MiaPaCa2 cellinjections, in contrast, were well-tolerated. Lung tissues of these micewere analyzed in IHC analysis at different time points after injection.In mice challenged with MiaPaCa-2 cells no metastases were detected inthe lungs after up to 73 days, and therefore this cell line was notselected as an IMAB362 treatment model. Suit-2 cancer cells metastasizedinto the lungs of the mice. Multiple foci were detected throughout thetissue. Therefore, the lentivirally CLDN18.2 transduced Suit-2-LVT cellline was selected as a model system to analyze the effects of IMAB362treatment on formation of metastasis.

In addition to Suit-2 also the ability of Patu8988S cells endogenouslyexpressing CLDN18.2 to form metastasis was analyzed. Engraftment checkswere performed with 2 different cell numbers (1×10⁶, 2×10⁶) i.v.injected per mouse. Lungs and livers were isolated at different timepoints as indicated in Table 18. First, the different tissues obtainedwere analyzed using Q-PCR. Lungs and livers obtained up to day 70 wereanalyzed by amplifying human a-satellite DNA of chromosome 17. Theresults of the lungs show a clear increase in the percentage of humanDNA in mouse lungs over time, which was not dependent on the injectedcell number. By i.v. application of 1×10⁶ or 2×10⁶ cells 5.8% and 3.7%human DNA could be detected after 70 days, respectively (FIG. 19). Inlivers, hardly any human DNA was amplified. After 70 days the percentagewas slightly increased, but still below 0.005%.

To verify CLDN18.2 expression in the Patu8988S metastasis, lung tissueswere immunohistochemically stained using anti-human MHC class Iantibodies for detection of human cells in mouse tissue, as well as theanti-Claudin18 (Mid) antibody. MHC-I staining showed that clearmetastasis foci were detectable in mouse lung tissue sections, but notin liver sections (FIG. 20). Furthermore, the membranes of the cells inthese foci were stained with the anti-Claudin18 (Mid) antibody showingclear expression of the IMAB362 target protein in these cells.Therefore, in addition to the Suit2-LVT model, this endogenousmetastasis model was selected for IMAB362 treatment investigation.

Example 4 IMAB362-Mediated Cell Killing Effects

IMAB362 Crosslinking Induces Efficient Apoptosis

Antibody binding to a cell surface target may initiate aberrantsignalling resulting directly in cell death. Such signalling events maydepend on the target epitope, the valency of binding and whether bindingis associated with cross-linking of the target. For several CD20positive lymphoma cell lines, for example induction of apoptosis byrituximab is only observed under cross-linking conditions. Suchcross-linking may take place in vivo when high affinityFc-receptor-positive immune cells interact with antibody-coated tumorcells.

Cross-linking of IMAB362 induces direct apoptosis within 18-42 hours inhuman gastric cancer cells NUGC-4 and KATO-III as measured by the TUNELassay. The magnitude of apoptosis correlates with the dose of theantibody and level of target expression on the cancer cell. Treatmentwith gemcitabine leads to cell cycle arrest of tumor cells followed byapoptotic cell death. Apoptosis of gemcitabine treated pancreas tumorcells is shown in FIG. 21.

IMAB362-Mediated ADCC Activity Against Pancreatic Cancer Cells

IMAB362 is highly potent in recruiting and activatingFey-receptor-positive immune effector cells, such as natural killercells. Binding of IMAB362 to target cells, induces antibody-dependentcellular cytotoxicity (ADCC) by granzymes and perforins secreted by theeffector cells upon binding of their Fey receptors to the antibody. Theimpact of this mechanism of action was previously shown for luciferase-and CLDN18.2-positive stomach CA cells (like NUGC-4 and KATO-III) byincubation with IMAB362 for 24 hours in the presence of human peripheralblood mononuclear cells (PBMC) (effector to target ratio=40:1).Application of up to 200 μs/mlIMAB362 resulted in maximum lysis rates of80-100%.

Here, we determined the ADCC activity of IMAB362 against pancreaticcancer cell lines. Increasing concentrations of IMAB362 were incubatedwith the different cell lines at an E:T ratio of 40:1. PBMCs ofdifferent donors were added in each experiment. Results for all celllines are summarized in Table 19. Of the 5 initially identifiedCLDN18.2-positive pancreas cell lines, only Patu8988S, Panc05.04 andDANG were efficiently killed by addition of IMAB362 and PBMCs (FIG.22A). Although CLDN18.2 surface expression was not detectable in FACSfor Panc05.04 and DANG, the expression level is significant enough tocause effector cell-dependent killing (EC₅₀: Patu8988S: 0.01-1.4 μg/ml,DANG/Pan05.04: 0.1-38 μg/ml). From these data it can be concluded thatonly cells expressing relative RNA levels>5.5×10⁵ are efficiently lysed.

ADCC analyses were also performed with LVT pancreatic cancer cell linesand their corresponding parental cell lines (FIG. 22B-F). ADCC strictlydepends on the specific binding of IMAB362 to the target, since onlyCLDN18.2 positive target cells are killed by IMAB362 and PBMCs. Halfmaximum killing and maximum killing rates induced in human pancreaticcancer cells by IMAB362 varied between PBMC donors and were alsodependent on passage number of the cells affecting CLDN18.2 expressionlevel.

The IMAB362 concentrations causing half maximum killing rates of thetarget cells as well as the maximum killing rates are shown in FIG.22G-H. The LVT pancreas CA cell lines were killed upon addition of smallamounts of antibody at high rates, whereas for DANG and Panc05 highestantibody concentrations are required to reach a ˜50% maximum killingrate. For Panc05.04 results obtained with subclone 15D3 (CLDN18.2positive clone selected by limited dilution of Panc05.04 and FACS) areincluded in the figure showing comparable ADCC lysis rates as with theLVT cell lines. Unfortunately, CLDN18.2 expression in this clone isquickly silenced in vitro after subculturing the cells and thus thisclone was not used for further experiments.

IMAB362-Mediated CDC Activity Against Pancreatic Cancer Cells

Pancreatic cancer cells, which were killed with IMAB362 in ADCC assays,were analyzed for their sensitivity towards the complement-dependentlytic activity of IMAB362. In addition, the LVT cell lines and theparental strains were tested in CDC.

CDC activity is activated by complexes of antigen and IgM or IgGantibodies (classical pathway) or by mircrobial surfaces (alternativepathway). In the classical pathway complement C5 is converted to C5b.The anaphyloatoxins C3a, C4a and C5b are released and a membrane attackcomplex (MAC) is formed by the sequential binding of C5b, C7, C8 and C9.This pathway is inhibited by soluble but also membrane bound proteins(e.g. CR1, DAF, MCP, CD59, CD55, CD46) protect self-tissues.

CHO-K1 cells stably transfected with CLDN18.2 (p740) and luciferase wereused as assay positive controls in each assay (FIG. 23A). The cell linesDANG, BxPC3, YAPC, Patu8988S, Panc05.04, CAPAN1 and Suit2 were not lysedby IMAB362 and addition of healthy human serum pool (FIG. 23B). AlthoughDANG, Patu8988S and Panc05.04 cells are CLDN18.2 positive as shown inall the previous experiments, these cells were not lysed in acomplement-dependent manner. This is most likely due to the fact thatneoplastic cells over-express one or more membrane bound complementinhibitory proteins (e.g. CD46, CD55 and CD59) (Geis et al., Curr CancerDrug Targets, 2010 10:922-931). However, if expression of theseinhibitory proteins on tumor cells affect clinical outcome of anantibody therapy is still contradictory (Dzietczenia et al. Med. Oncol.2010, 27:743-6; Weng and Levy at al., Blood 2001 98:1352-7). In additionto the endogenous cell lines, all the LVT cell lines were tested in CDCassays. As shown in FIG. 23, IMAB362 and serum addition toMiaPaCa-2-LVT, Suit2-LVT and CAPAN1-LVT resulted in dose-dependent lysiswith EC₅₀ values ranging from 0.3 to 2.6 μg/ml.

Overview of CLDN18.2 Expression in Human Pancreatic Cancer Cell Lines

TABLE 19 In house in vitro and in vivo characteristics of pancreascancer cell lines. CLDN18.2 CLDN18 CLDN18 CLDN18 mRNA Protein surfacecellular ADCC⁴ CDC level level expression localization Max. lysis EC₅₀Max. lysis EC₅₀ Selected for IMAB362 treatment Cell line (RT-PCR) (WB)¹(FACS)² (IF³) (%) (ng/ml) (%) (ng/ml) studies AsPC1   2E1 − 0.14%Cytoplasm n.m. n.m. n.d. n.d. No nuclear dots BxPC3 4.3E4 − 1.16% n.d.n.m. n.m n.d. n.d. No (ATCC) BxPC3 2.9E4 + 0.90% cytoplasm n.m.-60 n.mn.d. n.d. Cells able to engraft, used after LVT (ECACC) for in vivotreatments BxPC3 6.1E7 +++ cytoplasm + 90.6 ± 6.4  47.7 ± 19.1 n.m. n.m.Cells able to engraft, used for in vivo LVT membrane treatments CAPAN18.0E2 − 0.54% negative² n.m. n.m. n.m. n.m. Cells able to engraft, usedafter LVT for in vivo treatments CAPAN1- 1.5E5 +++ cytoplasm 92.6 ± 5.5 65.03 ± 79.1 970.1 Cells able to engraft, used for in vivo LVT membrane35.1 treatments CFPAC1 1.5E4 − 0.55% negative 0-1.5 n.m. n.d. n.d. NoDANG 4.9E5 ++ 1.19% cytoplasm 52.5 ± 7.8  4976.5 ± n.m. n.m. Cells ableto engraft, used for in vivo nuclear dots 4125.4 treatments(disadvantage: cachexia) DANG- 1.8E8 +++ cytoplasm + 85.9 ± 11.7 52.3 ±27.7 n.m. n.m. Cells able to engraft, used for in vivo LVT membranetreatments HPAFII 4.2E3 − 10.00% negative n.m. n.m. n.d. n.d. Selectionof single clones unsuccessful (disadvantage: cachexia) HPAC 3.6E1 −0.81% 0-22.2 n.m-495.6 n.d. n.d. Cells able to engraft, used after LVTfor in vivo treatments HPAC- 7.0E5 +++ cytoplasm + 76.2 ± 19.4 116.8 ±n.m. n.m. Cells able to engraft, used for in vivo LVT membrane 105.4treatments HUP-T3 1.3E4 − 0.23% nuclear dots n.m. 17.7 n.m. n.d. n.d. noHUP-T4 1.1E2 − 5.30% nuclear dots n.m. n.m. n.d. n.d. no KCI-MOH n.d. −n.d. negative² n.m.-2.8 n.m. n.d. n.d. no KP-2 1 − 0.54% negative n.d.n.d. n.d. n.d. no KP-4 n.d. − n.d. n.d. n.d. n.d. n.d. n.d. no MiaPaCa20 − 0.22% negative n.m. 8.1 n.m. n.m. n.m. Cells able to engraft, usedafter LVT for in vivo treatments MiaPaCa2- 1.7E8 +++ cytoplasm + 78.1 ±6.0  23.4 ± 12.3 78.4 340 Cells able to engraft, used for in vivo LVTmembrane treatments Panc01 5.3E1 −   65% nuclear dots 0-4.5 n.m. n.d.n.d. no Panc02.03 n.d. − n.d. negative² n.m. n.m. n.d. n.d. no Panc03.272.7E5 + 7.30% cytoplasm n.m. n.m. n.d. n.d. Cells engraft, some CLDN18.2staining observed Panc04.03 n.d. − 1.38% negative² n.m.-41.4 n.m. n.d.n.d. No Panc05.04 1.2E5 ++   15% cytoplasm + 67.1 ± 6.8  5728.8 ± n.m.n.m. Selection of single clones membrane 4435.1 unsuccessfull. Cells notable to engraft (standard procedure). Patu8902 1.7E3 − 0.39% negativen.m.-4.2 n.m. n.m. n.m. Cells able to engraft, used after LVT forengrafments Patu8902- 1.6E8 +++ cytoplasm + 74.1 ± 16.4 49.2 ± 86.2 76.7291.0 Cells able to engraft s.c., used for LVT membrane metastasisengraftments, mice die upon i.v. application Patu8988S 3.9E5 +++ 81.70%cytoplasm + 70.1 ± 22.1 174.8 ± n.m. n.m. Cells able to engraft, formmembrane 137.9 heterogeous tumors/cysts, used for in vivo treatments(for s.c. tumors and metastasis) Patu8988T 1.5E1 − 6.00% nuclear dotsn.m.-27.54 n.m-470.5 n.d. n.d. No Suit2 1.0E4 − 0.41% negative n.m. n.m.n.m. n.m. Cells able to engraft, used after LVT for metastasis studySuit2-LVT 2.0E8 +++ cytoplasm + 81.2 ± 7.6  73.2 ± 14.7 n.m. n.m. Cellsable to engraft s.c., used for membrane metastasis assay Su86.86 3.2E4 −1.51% negative n.m.-14.8 n.m.-30.4 n.d. n.d. no SW1990 1.8E1 − 2.53%negative n.m. n.m. n.d. n.d. no YPAC 1.5E5 (+) 0.23% negative n.m. n.m.n.m. n.m. Cells able to engraft, used after LVT for in vivo treatmentsYAPC- 2.5E8 +++ cytoplasm + 91.5 ± 6.3  29.5 ± 15.7 n.m. n.m. Cells ableto engraft, used for in vivo LVT membrane treatments ¹usinganti-Claudin18 (C-term) antibody (Zymed) ²staining of 2E5 cells/100 μlwith 50 μg/ml IMAB362 ³using antibody 35-22A on fixed and permeabilizedcells ⁴Results obtained with at least 2 donors; n.m.: not measureable;n.d. not determined.

Example 5 Efficacy of IMAB362 on Pancreatic Cancer Xenograft Models

10 of the 41 tested pancreatic cancer xenograft models were chosen toinvestigate the efficacy of IMAB362 in vivo. Using pancreatic xenograftmodels with high expression of CLDN18.2, IMAB362 treatment showed a highantitumoral effect. This was investigated by treatment of mice bearingBxPC3-LVT or MiaPaCa-2-LVT xenografts subcutaneous in the left flank.Treatment was initiated 3 days after tumor inoculation with injectionsof 200 μg IMAB362 semi-weekly. The IMAB362 treated mice showedsignificantly inhibited tumor growth compared to mice treated withsaline control. In addition tumor growth suppression of IMAB362 treatedmice resulted in prolonged median survival (FIG. 24 and FIG. 25).IMAB362 efficacy correlates with the duration of treatment. Initiationof IMAB362 treatment at early time points had an increased effect ontumor growth inhibition than late treatment starts to examine effect onestablished tumors. Furthermore the antitumoral effect of IMAB362depended on the amount of CLDN18.2 target expression. IMAB362 mediatedgrowth inhibition of low CLDN18.2 expressing tumors like DANG andPatu8988S xenografts was reduced compared to inhibition of tumor growthusing high CLDN18.2 expressing xenograft tumors.

Example 6 Treatment of Pancreatic Metastasis Mouse Models

TABLE 20 Summary of treatments for testing IMAB362 efficacy onpancreatic cancer metastasis Experiment No. Treatment Cell line (Date)groups Experimental set-up Suit2-LVT ET2_C220 1. n = 15; 200 μg IMAB362Injection of 2e6 cells i.v. into (22.11.2011) semi-weekly i.v./i.p. thetail vein of female Hsd: 2. n = 15; 200 μg isotype AthymicNude-Foxn1^(nu) mice control semi-weekly Initiation of treatment 3 daysi.v./i.p. after tumor cell injection 3. n = 15; PBS semi- weeklyi.v./i.p. Patu8988S ET1_C178 1. n = 10; 200 μg IMAB362 Injection of 2e6cells i.v. into (24.05.2011) semi-weekly i.v./i.p. the tail vein offemale Hsd: 2. n = 10; PBS semi- Athymic Nude-Foxn1^(nu) mice weeklyi.v./i.p. Initiation of treatment 3 days after tumor cell injectionPatu8988S ET1_C178b 1. n = 15; 200 μg IMAB362 Injection of 2e6 cellsi.v. into (03.06.2011) semi-weekly i.v./i.p. the tail vein of femaleHsd: 2. n = 15; isotype control Athymic Nude-Foxn1^(nu) mice semi-weeklyi.v./i.p. Initiation of treatment 3 days after tumor cell injection

Suit2-LVT Metastasis Model:

Mice were intravenously injected with 2×10⁶ Suit2-LVT cells and weretreated with 200 μg IMAB362, isotype control antibody (IMAB027), or withPBS as indicated in Table 20. After 35 days the first mouse (isotypecontrol group) died. Consequently, all mice were sacrificed on day 42and lungs and livers were taken for IHC and Q-PCR analyses.

Q-PCR analysis of human DNA in the lungs of mice was repeated at leasttwice in triplicate. The calculation of the percentage of human DNA withthe obtained Ct values revealed a significant decrease (P<0.05) inSuit2-LVT metastases detected in the lungs, if mice were treated withIMAB362 (FIG. 26A) as compared to both PBS and isotype controltreatments. To confirm these results, tissue sections of the lungsamples were prepared and stained using MHC-I antibodies. The surface ofthe positively stained cells in the lung sections was calculated usingthe ImageJ Program. For IMAB362 treatment significant inhibition(P<0.05) was observed as compared to PBS treatment confirming theresults obtained with Q-PCR. For the isotype control antibody however,the differences were not significant (FIG. 26B). This discrepancy ismost likely due to differences in tissue processing: IHC processing oftissue sections provides only insight into a very small section of thelung compared to Q-PCR analysis, for which the genomic DNA is extractedfrom half of the tissue.

In addition to tissue processing, it is possible that the resultrepresents unexpected inhibiting effects of the isotype control antibodytargeting CLDN6. To investigate this option, the Suit2-LVT cells wereanalyzed for CLDN6 expression and IMAB027 binding in FACS. The additionof 200 μg/ml IMAB362 to Suit2-LVT cells confirmed strong binding to thecells, whereas addition of 200 μg/ml IMAB027 resulted in weak binding ofthe antibody to these target cells, indicating that CLDN6 is indeedweakly expressed on these cells. These results suggest that at least twofactors (tissue processing and weak IMAB027 inhibition) resulted in theobserved discrepancies with the isotype control antibody.

Patu8988S Metastasis Model

To analyze the effect of IMAB362 treatment on the development and growthof Patu8988S metastasis in vivo, 10 mice per group were injected with2×10⁶ Patu8988S cells. The first experiment was performed by comparingIMAB362 treatment to PBS treated mice. In each group 1 mouse diedimmediately after injection of the cells. In the other 18 mice, themetastasis developed very quickly as compared to the engraftmentexperiments. After 63 days the first 2 mice in the PBS group weresacrificed due to bad health conditions. All other mice were sacrificedafter 65 days. The optical analysis of the lungs revealed largemetastasis throughout the lung tissues. The amount of metastasis wasanalyzed in Q-PCR experiments (FIG. 27). The results show that IMAB362inhibits the growth of metastasis in lung tissue.

A second experiment with 11 mice per group was performed by comparingIMAB362 treatment with isotype control (Rituximab) control treatment. Inthis experiment metastasis developed slowly as observed in theengraftments. Nevertheless, for comparability this second experiment wasterminated after 65 days. Again lung tissues were analyzed in Q-PCR andagain IMAB362 reduced the growth of the metastasis. One mouse of theIMAB362 group was identified as outlier and exclusion of this outlierresulted in almost significant (P=0.0588) inhibition. These data wereverified by IHC surface analysis as described for the Suit2-LVTmetastasis experiment. Here the same outlier could be identified, andomitting this value in the t-test, the inhibition of IMAB362 is also atthe border of being significant (P=0.0691), that values of the samemouse.

Example 7 Primary Pharmacodynamics of IMAB362 in Combination withChemotherapy

Sensitivity of pancreatic carcinoma cells to gemcitabine and oxaliplatinPancreatic cancer cell lines constitutively expressing CLDN18.2 (DANG,Patu8988S) and cells stably transduced with CLDN18.2 (MiaPaCa-2-LVT,BxPC3-LVT) were used to investigate modes of action of IMAB362 incombination with the chemotherapeutic agents oxaliplatin or gemcitabine.

Chemically, gemcitabine (Gemzar, marketed by Eli Lilly&Co) is anucleoside analog. As with 5-fluorouracil (5-FU) and other analogues ofpyrimidines, the triphosphate analogue of gemcitabine replaces one ofthe building blocks of nucleic acids during DNA replication. The processarrests tumor growth, as only one additional nucleoside can be attachedto the “faulty” nucleoside, resulting in apoptosis.

Oxaliplatin functions by forming both inter- and intra-strand crosslinks in DNA. Cross links in DNA prevent DNA replication andtranscription, resulting in cell death. (Graham, Joanne; Mushin,Mohamed; Kirkpatrick, Peter (January 2004). “Oxaliplatin”. NatureReviews Drug Discovery 3 (1): 11-2.)

Dose response curves of gemcitabine and oxaliplatin showed differentsensitivities of tested pancreatic tumor cell lines (FIG. 28 and FIG.29).

TABLE 21 IC50 values of gemcitabine and oxaliplatin for pancreaticcarcinoma cell lines. IC50 Inhibition of Proliferation Gemcitabine OxaliBxPC3-LVT ~2 ng/ml 500-1000 ng/ml Capan1-LVT ~1 ng/ml ~100 ng/ml DANG ~1ng/ml ~500 ng/ml Patu8988S-luci#6 >100 ng/ml >500 ng/ml MiaPaCa-2-LVT 50ng/ml 50-100 ng/ml

To inhibit cell proliferation of Patu8988S high concentrations ofgemcitabine (IC50>100 ng/ml) or oxaliplatin (IC50>500 ng/ml) arenecessary. DANG and BxPC3-LVT reacts very sensitive to gemcitabine butnot to oxaliplatin. MiaPaCa-2-LVT cells react most sensitive tooxaliplatin but less sensitive to treatment with gemcitabine (FIG. 28,FIG. 29 and Table 21).

Effect of Chemotherapeutic Agents on CLDN18.2 Expression in PancreaticCarcinoma Cell Lines

Mode of action triggered by IMAB362 binding depends strictly on thepresence and cell surface density of its target CLDN18.2. Pretreatmentof DANG and Patu8988S cells with gemcitabine (Gem) as well asgemcitabine in combination with oxaliplatin (GemOx) resulted inincreased mRNA and protein levels of CLDN18.2 shown by RT-PCR (FIG. 30)and western blot (FIG. 31) analysis of untreated and chemotherapypretreated cells. Consequently, the amount of CLDN18.2 proteintargetable by IMAB362 on the surface of Gem or GemOx pretreatedpancreatic cancer cell lines was increased as shown by flow cytometry(FIG. 32).

Treatment of DANG and Patu8988S with gemcitabine leads to upregulationof CLDN18.2. Patu8988S show strong upregulation of CLDN18.2 with Gem andlower upregulation with GemOx.

Effect of Chemotherapeutic Compounds on Cell Cycle and CLDN18.2Expression

Cell cycle progression refers to the sequence of events between onemitotic division and another in a cell. A resting phase (G0/G1) isfollowed by a DNA synthesis phase (S), then by a phase of cellenlargement (G2) and DNA replication (M) is followed by a division ofthe cell into two progeny cells. Any interference with the cellmachinery may inhibit all cycle progression at any phase of the cellcycle. For example, specific chemotherapeutic agents may blockprogression in either G2 or M or in both G2 an M (G2/M).

Gemcitabine treatment of DANG or Patu8988S leads to cell cycle arrest inS-Phase (FIG. 33, FIG. 34). Patu8988S cultivated with Gem were analysed.Gemcitabine treatment not only leads to cell cycle arrest it alsochanges expression of CLDN18.2 (FIG. 34B). The change of CLDN18.2density after gemcitabine treatment is even higher when comparingproliferating cells in S phase to resting cells in G0/G1 phase (FIG.34C). In Patu8988S cells, CLDN18.2 is expressed in all phases of thecell cycle. Upon treatment with gemcitabine, its expression is evenincreased, with the highest levels of CLDN18.2 per cell being found inthe S phase cell population.

This perturbation of tumour cell phenotype has a significant impact onthe biological effectiveness of therapeutic antibodies. ADCC and CDC aredose-related and therefore an increase of the target structure CLDN18.2provides synergistic benefit to standard chemotherapeutic regimes.

Kato III cells, a human gastric tumor cell line, was cultivated in RPMI1640 medium (Invitrogen) containing 20% FCS (Perbio) and 2 mM Glutamax(Invitrogen) at 37° C. and 5% CO₂, with or without cytostatic compounds.5-FU (Neofluor from NeoCorp AG) was tested at a concentration of 10 or100 ng/ml, and oxaliplatin (Hospira) was tested at a concentration of 50or 500 ng/ml. 8×10⁵ Kato III cells were cultivated for 96 hours withoutmedium change or for 72 hours followed by 24 hours cultivation instandard medium to release cells from cell cycle arrest in a E-welltissue culture plate at 37° C., 5% CO₂. Cells were harvested withEDTA/trypsin, washed and analysed.

For extracellular detection of CLDN18.2 cells were stained with themonoclonal anti-CLDN18.2 antibody IMAB362 (Ganymed) or an isotyp-matchedcontrol antibody (Ganymed). As secondary reagent goat-anti-huIgG-APCfrom Dianova was used.

Cell cycle stages were determined based on measurement of cellular DNAcontent. This allows one to discriminate between cells in the G1-, S- orG2-phase of the cell cycle. In the S-phase DNA duplication occurswhereas in the G2-phase cells grow and prepare for mitosis. Cell cycleanalysis was done using the CycleTEST PLUS DNA Reagent Kit from BDBiosciences following the manufacturer's protocol. Flow cytometryacquisition and analysis were performed by using BD FACS CantoII (BDBiosciences) and FlowJo (Tree Star) software.

The columns in FIGS. 35 a and b show the respective percentage of cellsin the G1-, S- or G2-phase of the cell cycle. Medium cultivated Kato IIIcells show a cell cycle arrest predominantly in the G1-phase. Cellstreated with 5-FU are blocked predominantly in the S-phase. Oxaliplatintreated Kato III cells show enrichment of cells predominantly in the G1-and G2-phases. As can be seen in FIG. 35 c, a cell cycle arrest in theS-phase or G2-phase results in stabilization or upregulation ofCLDN18.2. As soon as cells are released from any phase of the cell cycle(FIG. 35 b) the expression of CLDN18.2 on the cell surface of Kato IIIcells is upregulated (FIG. 35 d).

Kato III cells were pretreated for 4 days with Irinotecan or Docetaxeland analysed for CLDN18.2 expression and cell cycle arrest. Treatment ofcells with Irinotecan resulted in a dose dependent inhibition of cellgrowth and a cell cycle arrest in the S/G2-phase (FIG. 36). Treatment ofcells with Docetaxel resulted in a dose dependent inhibition of cellgrowth and a cell cycle arrest in the G2-phase (FIG. 36).

Effect of Chemotherapy on IMAB362 Induced Antibody Dependent CellularCytotoxicity (ADCC)

A series of experiments were performed with constitutively CLDN18.2expressing pancreatic cancer cell lines Patu8988S and DANG. Toinvestigate the effects of gemcitabine (Gem) or gemcitabine+oxaliplatin(GemOx) on IMAB362-mediated ADCC. Dose-response curves for IMAB362mediated cell lyses of pretreated cells were compared with mediumcultivated.

Dose response curves of Gem (1 ng/ml) or GemOx (Gem 1 ng/ml+Ox 10 ng/ml)pretreated DANG (2 days) are shifted upwards and to the left compared tountreated target cells (FIG. 37A). Treatment of tumor cells with Gem orGemOx leads to upregulation of CLDN18.2 and higher susceptibility forIMAB362 mediated ADCC. We could observe a decrease of the EC50 valuesand higher maximal cell lyses for IMAB362-mediated ADCC (FIG. 37B) inDANG cells after treatment with chemotherapeutic agents.

Peripheral blood mononuclear cells (PBMCs) including NK cells,monocytes, mononuclear cells or other effector cells from healthy humandonors were purified by Ficoll Hypaque density centrifugation. Washedeffector cells were seeded in X-Vivo medium. Kato III cells whichexpress CLDN18.2 endogenously and are of gastric origin were used astarget cells in this setting. Target cells stably expressed luciferase,lucifer yellow, which is oxidized by viable cells only. Purifiedanti-CLDN18.2 antibody IMAB362 was added at various concentrations andas an isotype control antibody an irrelevant chim huIgG1 antibody wasused. Samples were assayed for cytolysis by measuring luminescenceresulting from the oxidation of lucifer yellow which is a value for theamount of viable cells left after IMAB362 induced cytotoxicity. Kato IIIpretreated for 3 days with Irinotecan (1000 ng/ml), Docetaxel (5 ng/ml)or Cisplatin (2000 ng/ml) were compared to untreated medium cultivatedtarget cells and IMAB362 induced ADCC was quantified.

Kato III cells pretreated for 3 days with Irinotecan, Docetaxel orCisplatin exhibited a lower level of viable cells compared to mediumcultivated target cells (FIG. 38 a) and claudin18.2 expression in cellspretreated with Irinotecan, Docetaxel or Cisplatin was increasedcompared to medium cultivated cells (FIG. 38 b).

Furthermore, pretreatment of Kato III cells with Irinotecan, Docetaxelor Cisplatin augmented the potency of IMAB362 to induce ADCC (FIG. 38 c,d).

Effect of Chemotherapy on IMAB362 Induced CDC

The CDC potency of IMAB362 has been characterized by incubation withtarget cells in the presence of human serum as source of complement.

Medium cultivated MiaPaCa-2-LVT show EC50 values for IMAB362 specificlyses of 7665 ng/ml. Treatment with Gem leads to decrease of EC50 to4677 ng/ml compared with increase of max lyses (FIG. 39).

Effects of chemotherapeutic agents on IMAB362-induced CDC were analyzedby pretreating KATO III gastric cancer cells with 10 ng/ml 5-FU and 500ng/ml oxaliplatin (5-FU+OX) for 48 hours. Representative dose responsecurves of IMAB362-induced CDC using chemotherapeutic pretreated KATO IIIcells are shown in FIG. 40. Pretreatment of tumor cells for 48 hoursaugmented the potency of IMAB362 to induce CDC, resulting in highermaximal cell lysis of pretreated tumor cells compared to untreatedcells.

Example 8 Efficacy of IMAB362 in Combination with Chemotherapy in MouseTumor Models

Antitumoral activity of IMAB362 in combination with Gem or GemOx wasexamined in subcutaneous pancreatic carcinoma xenograft models, whichwere used for testing efficacy of IMAB362 as single agent before.

BxPC3-LVT or MiaPaCa-2-LVT tumor bearing nude mice treated with IMAB362showed significant tumor growth retardation compared to control micetreated with saline control. Chemotherapy with up to 100 mg/kggemcitabine without additional IMAB362 treatment showed no significanttherapeutic effect on BxPC3-LVT or MiaPaCa-2-LVT xenograft. In contrast,the combined treatment with 50-100 mg/kg gemcitabine plus IMAB362resulted in significantly increased tumor growth inhibition and inprolonged survival of tumor bearing mice compared to mice treated withchemotherapy alone (FIG. 41, FIG. 42, FIG. 43). These observationsindicate the existence of synergistic therapeutic effects by combinationof gemcitabine and IMAB362 immunotherapy.

When using high doses of gemcitabine with 2×150 mg/kg per week,established MiaPaCa-2-LVT xenograft tumors are strongly inhibited intumor growth independent from IMAB362 treatment (FIG. 44A). However,mice treated with combined therapy of IMAB362 and gemcitabine showedhighly significant prolonged survival compared to mice treated withgemcitabine as single agent (FIG. 44B).

Example 9 ZA/IL-2 Treatment Results in Expansion of High Amounts ofVγ9Vδ2 T-Cells

PBMCs were cultivated for 14 days in RPMI medium supplemented with 300U/ml IL-2 and with or w/o 1 μM zoledronic acid (ZA). The percentage ofVγ9+Vδ2+ T cells within the CD3+ lymphocyte population and thepercentage of CD16+ cells within the CD3+Vγ9+Vδ2+ T cell population wasdetermined by multicolor FACS on day 0 and day 14.

IL-2 addition in the PBMC cultures is required for survival and growthof lymphocytes. They efficiently expand in cultures supplied with 300U/ml IL-2. FACS analysis using Vγ9 and Vδ2 specific antibodies revealthat addition of ZA/IL-2 specifically induces the accumulation of Vγ9Vδ2T cells. After 14 days, the CD3+ lymphocyte population can comprise upto 80% of Vγ9Vδ2 T cells. A portion of Vγ9Vδ2 T cells express CD16,whereas enrichment of these cells within the CD3+ lymphocyte populationis 10-700 fold, dependent on the donor. Enrichment of the CD16+Vγ9+Vδ2+T cells in the cultures is 10-600 fold higher as compared to culturesgrown without ZA. We conclude that ZA/IL-2 treatment of PBMCs in vitroresults in the up-regulation of the ADCC-mediating FcγIII receptor CD16in a significant proportion of γδ T cells.

Similar to NK cells, the ZA/IL-2 expanded Vγ9Vδ2 T cells are positivefor CD16, the FcγRIII receptor via which a cell-bound antibody triggersADCC. To evaluate whether Vγ9Vδ2 T cells are capable of inducing potentADCC in conjunction with IMAB362 a series of experiments has beenperformed.

PBMCs derived from 2 different donors (#1 and #2) were cultivated inmedium with 300 U/ml IL-2 and with or w/o 1 μM ZA. After 14 days cellswere harvested and added with increasing concentrations (0.26 ng/ml-200μg/ml) of IMAB362 to NUGC-4 cells expressing CLDN18.2. Specific killingwas determined in luciferase assays. ADCC assays were performed with 27donors grown in 300 U/ml IL-2 and either with or w/o ZA wherein NUGC-4served as target cells. For each donor, the EC₅₀ values calculated fromthe dose-response curves and the maximum specific killing rate at a doseof 200 μg/ml IMAB362 were scored in the scatter plots.

Strong IMAB362-dependent ADCC activity was observed againstCLDN18.2-positive NUGC-4 cells using PBMCs cultivated for 14 days withZA/IL-2. Using ZA/IL-2-treated PBMC cultures, ADCC depends on thepresence of Vγ9 Vδ2 T cells. If cells are cultured without ZA, ADCCactivity is reduced for most donors. In these cultures, residual ADCCactivity is NK-cell dependent. By testing more than 20 donors, ADCCassays reveal that ZA/IL-2 treatment of PBMCs improves the EC₅₀ andmaximum specific killing rates as compared to PBMCs cultured with IL-2alone.

Example 10 Efficacy of IMAB362 in Combination with Gemcitabine in MouseMetastasis Model

To analyze the effect of IMAB362 in combination with gemcitabinetreatment on Patu8988S lung metastases in vivo, 12 Hsd:AthymicNude-Foxn1^(nu) mice per group were treated with an intravenousinjection 2×10⁶ Patu8988S cells into the tail vein. 14 days post tumorcell injection mice were treated with 200 μg IMAB362 or PBS as control(i.v./i.p.) semi-weekly plus a weekly dose 100 mg/kg gemcitabine i.p.for 4 weeks. Treatment with IMAB362 or PBS was maintained until micewere sacrificed on day 70 post tumor cell injection. Analysis of thexenografted tumor load in the lungs was performed by QPCR of human DNAin the prepared lungs and by an optical analysis of animmunohistological staining with an anti-human MHC-I antibody (cloneEPR1394Y). The results show that mice treated with IMAB362 plusgemcitabine have a significantly reduced amount of human DNA in theirlungs (FIG. 45A) and that the surface of lung slices stained againsthuman MHC-I complex is significantly smaller than in lungs of micetreated with an irrelevant antibody plus gemcitabine (FIG. 45B). Bothmethods reveal a reduced tumor load of Patu8988s xenografts in the lungsof IMAB362 plus gemcitabine treated mice, showing that the combinationwith IMAB362 is significantly superior to a monotherapy withgemcitabine.

1. A method of treating or preventing pancreatic cancer in a patientcomprising administering to the patient (i) an antibody having theability of binding to CLDN18.2 and (ii) an agent stabilizing orincreasing expression of CLDN18.2.
 2. The method of claim 1, whereinexpression of CLDN18.2 is at the cell surface of a cancer cell.
 3. Themethod of claim 1 or 2, wherein the agent stabilizing or increasingexpression of CLDN18.2 comprises an agent which induces a cell cyclearrest or an accumulation of cells in one or more phases of the cellcycle, preferably in one or more phases of the cell cycle other than theG1-phase, more preferably in the G2-phase and/or the S-phase.
 4. Themethod of claim 1, wherein the agent stabilizing or increasingexpression of CLDN18.2 comprises an agent selected from the groupconsisting of nucleoside analogs, platinum compounds, camptothecinanalogs, taxanes, prodrugs thereof, salts thereof, and combinationsthereof.
 5. The method of claim 1, wherein the agent stabilizing orincreasing expression of CLDN18.2 comprises an agent selected from thegroup consisting of gemcitabine, 5-fluorouracil, oxaliplatin,irinotecan, paclitaxel, prodrugs thereof, salts thereof, andcombinations thereof.
 6. The method of claim 1, wherein the agentstabilizing or increasing expression of CLDN18.2 comprises an agentinducing immunogenic cell death.
 7. The method of claim 6, wherein theagent inducing immunogenic cell death comprises oxaliplatin.
 8. Themethod of claim 1, wherein the method comprises administering acombination of gemcitabine and oxaliplatin, a combination of gemcitabineand cisplatin, a combination of gemcitabine and carboplatin or acombination of oxaliplatin, 5-fluorouracil or prodrugs thereof andirinotecan.
 9. The method of claim 1, wherein the method comprisesadministering folinic acid, oxaliplatin, 5-fluorouracil or prodrugsthereof and irinotecan.
 10. A method of treating or preventing cancer ina patient comprising administering to the patient (i) an antibody havingthe ability of binding to CLDN18.2 and (ii) gemcitabine.
 11. The methodof claim 10, wherein the cancer is pancreatic cancer.
 12. The method ofclaim 1, wherein the method further comprises administering an agentstimulating γδ T cells, wherein the γδ T cells are preferably Vγ9Vδ2 Tcells.
 13. The method of claim 12, wherein the agent stimulating γδ Tcells is a bisphosphonate.
 14. The method of claim 12 or 13, wherein theagent stimulating γδ T cells is a nitrogen-containing bisphosphonate(aminobisphosphonate).
 15. The method of claim 12, wherein the agentstimulating γδ T cells is selected from the group consisting ofzoledronic acid, clodronic acid, ibandronic acid, pamidronic acid,risedronic acid, minodronic acid, olpadronic acid, alendronic acid,incadronic acid and salts thereof.
 16. The method of claim 12, whereinthe agent stimulating γδ T cells is administered in combination withinterleukin-2.
 17. The method of claim 1, wherein the antibody havingthe ability of binding to CLDN18.2 binds to the first extracellular loopof CLDN18.2.
 18. The method of claim 1, wherein the antibody having theability of binding to CLDN18.2 mediates cell killing by one or more ofcomplement dependent cytotoxicity (CDC) mediated lysis, antibodydependent cellular cytotoxicity (ADCC) mediated lysis, induction ofapoptosis and inhibition of proliferation.
 19. The method of claim 1,wherein the antibody having the ability of binding to CLDN18.2 is anantibody selected from the group consisting of (i) an antibody producedby and/or obtainable from a clone deposited under the accession no. DSMACC2737, DSM ACC2738, DSM ACC2739, DSM ACC2740, DSM ACC2741, DSMACC2742, DSM ACC2743, DSM ACC2745, DSM ACC2746, DSM ACC2747, DSMACC2748, DSM ACC2808, DSM ACC2809, or DSM ACC2810, (ii) an antibodywhich is a chimerized or humanized form of the antibody under (i), (iii)an antibody having the specificity of the antibody under (i) and (iv) anantibody comprising the antigen binding portion or antigen binding site,in particular the variable region, of the antibody under (i) andpreferably having the specificity of the antibody under (i).
 20. Themethod of claim 1, wherein the method comprises administering theantibody having the ability of binding to CLDN18.2 at a dose of up to1000 mg/m².
 21. The method of claim 1, wherein the method comprisesadministering the antibody having the ability of binding to CLDN18.2repeatedly at a dose of 300 to 600 mg/m².
 22. The method of claim 1,wherein the cancer is CLDN18.2 positive.
 23. The method of claim 1,wherein CLDN18.2 has the amino acid sequence according to SEQ ID NO: 1.24. The method of claim 1, wherein the pancreatic cancer comprisesprimary cancer, advanced cancer or metastatic cancer, or a combinationthereof such as a combination of pancreatic primary cancer andmetastatic cancer.
 25. The method of claim 24, wherein the metastaticcancer comprises metastasis to the lymph nodes, ovary, liver or lung, ora combination thereof.
 26. The method of claim 1, wherein the pancreaticcancer comprises a cancer of the pancreatic duct.
 27. The method ofclaim 1, wherein the pancreatic cancer comprises an adenocarcinoma orcarcinoma, or a combination thereof.
 28. The method of claim 1, whereinthe pancreatic cancer comprises a ductal adenocarcinoma, a mucinousadenocarcinoma, a neuroendocrine carcinoma or an acinic cell carcinoma,or a combination thereof.
 29. The method of claim 1, wherein thepancreatic cancer is partially or completely refractory to gemcitabinetreatment such as gemcitabine monotherapy.
 30. The method of claim 1,wherein preventing pancreatic cancer comprises preventing a recurrenceof pancreatic cancer.
 31. The method of claim 1, wherein the patient hada surgery for pancreatic cancer.
 32. The method of claim 1, wherein thepatient has a precancerous pancreatic lesion, in particular aprecancerous pancreatic lesion comprising a beginning malignanthistological change in the pancreatic ducts.
 33. A medical preparationfor treating or preventing pancreatic cancer comprising (i) an antibodyhaving the ability of binding to CLDN18.2 and (ii) an agent stabilizingor increasing expression of CLDN18.2.
 34. The medical preparation ofclaim 33, which is present in the form of a kit comprising a firstcontainer including the antibody having the ability of binding toCLDN18.2 and a second container including the agent stabilizing orincreasing expression of CLDN18.2.
 35. The medical preparation of claim33 or 34, further including printed instructions for use of thepreparation for treatment or prevention of pancreatic cancer.
 36. Amedical preparation comprising (i) an antibody having the ability ofbinding to CLDN18.2 and (ii) gemcitabine.
 37. The medical preparation ofclaim 36 which is for treating or preventing cancer, in particularpancreatic cancer.
 38. The medical preparation of claim 36 or 37, whichis present in the form of a kit comprising a first container includingthe antibody having the ability of binding to CLDN18.2 and a secondcontainer including gemcitabine.
 39. The medical preparation of claim36, further including printed instructions for use of the preparationfor treatment or prevention of cancer, in particular pancreatic cancer.